Protein kinase inhibitors for treatment of neurological disorders

ABSTRACT

The invention features novel derivatives of K-252a, as well as novel bis-N-substituted derivatives of staurosporine. The invention also features a method for treating diseased neuronal cells involving the administration of either the novel staurosporine derivatives or specified functional derivatives of K-252a.

This application is a continuation-in-part of U.S. Ser. No. 08/329,540,filed Oct. 26, 1994, which is a continuation-in-part of U.S. Ser. No.08/096,561, filed Jul. 22, 1993 parented as U.S. Pat. No. 5,461,146,which is a continuation-in-part of U.S. Ser. No. 07/920,102, filed Jul.24, 1992, now abandoned.

BACKGROUND OF THE INVENTION

Protein kinases are a broad class of enzymes which act to modifychemically many cellular proteins, by phosphorylation of amino acids.

Inhibitors of protein kinases are structurally varied, and have variable(and sometimes contradictory) effects on the nervous system and othertissues. A given protein kinase inhibitor may influence more than oneprotein kinase. For example, K-252a, an alkaloid-like material isolatedfrom the culture broth of Nocardiopsis sp. and Actinomadula sp. wasoriginally reported to be a protein kinase C inhibitor, but wassubsequently found also to inhibit protein kinases A and G, myosinlight-chain kinase, and trk (a tyrosine kinase activated by nerve growthfactor NGF!, the latter a neurotrophic protein which promotes thesurvival of peripheral, sensory and sympathetic neurons).

Consistent with this latter effect, K-252a blocks the neurotrophicactions of NGF on PC-12 cells (chromaffin cells from rat adrenalmedullary tumors, pheochromocytomas), and promotes the survival ofdorsal root ganglion neurons and hippocampal neurons. However, it hasbeen found to be cytotoxic at a wide range of concentrations, leadingsome investigators to conclude that it has limited usefulness in vivo.

A microbial alkaloid related to K-252a, staurosporine, also has avariety of effects on different protein kinases and cell types.Staurosporine was found to have NGF-like effects on PC-12 cells, and toprotect the gerbil hippocampus from post-ischemic injury. It is able toreverse damage to cholinergic neurons in the rat basal forebrain.

K-252a and staurosporine have been proposed as tumor inhibitors.Staurosporine has been offered as an insecticide. Derivatives ofstaurosporine, with a hydrocarbyl radical or an acyl radical substitutedat the methylamine nitrogen, have been made and proposed for thefollowing uses: tumor inhibition, inflammation inhibition,immunomodulation, and treatment of diseases of the cardiovascular andcentral nervous systems.

SUMMARY OF THE INVENTION

The invention features, in one aspect, novel bis-N-substitutedderivatives of staurosporine, represented by the formula:

     Stau!-N(CH.sub.3)--W--N(CH.sub.3)- Stau!                  (I)

where Stau! represents a residue of the formula: ##STR1## and Wrepresents a bis(carbamyl) or bis(thiocarbamyl) radical,

    --C(═Y)--NH--W'--NH--C(═Y)--

where W' is a hydrocarbylene radical of 2-20 carbon atoms and Y is O orS.

In a preferred aspect the invention features, e.g., the compounds

1,6-hexamethylene-bis-(carbamylstaurosporine) (HBCS);

ρ-phenylene-bis-(carbamylstaurosporine) (PBCS);

The invention also features a novel derivative of K-252a, represented bythe formula (II-4): ##STR2## where R¹, R², Z¹ and Z² are eachindependently H; X is hydroxymethyl (CH₂ OH); and R is OCH₃.

The invention also features a novel derivative of K-252a, represented bythe formula: ##STR3## where R¹, R², Z¹ and Z² are each independently H;X is CH₂ --NH-SerH; and R is OH.

Also included in the invention are compounds represented by thefollowing Formula (II-49): ##STR4## wherein R², Z¹, and Z² are each H; Ris OH; R¹ is CH₂ SO₂ C₂ H₅ ; and X is CO₂ CH₃.

Also included in the invention are compounds represented by thefollowing Formula (II-38): ##STR5## wherein R¹, R², Z¹, and Z² are eachH; R is OH; and X is CH₂ NHCO₂ C₆ H₅.

Also included in the invention are compounds represented by thefollowing Formula (II-45): ##STR6## wherein R¹ and R² are each Br; R isOH; Z¹ and Z² are each H; and X is CONHC₆ H₅.

Also included in the invention are compounds represented by thefollowing Formula (II-57): ##STR7## wherein R¹, R², Z¹, and Z² are eachH, R is OH; and X is CH₂ NHCO₂ CH₃.

Also included in the invention are compounds represented by thefollowing Formula (II-72): ##STR8## wherein R¹ is CH₂ S(CH₂)₂ NH₂ ; X isCO₂ CH₃ ; R is OH; and R², Z¹, and Z² are each H.

Also included in the invention are compounds represented by thefollowing Formula (II-75): ##STR9## wherein R¹ is ##STR10## X is CO₂ CH₃; R is OH; and R², Z¹, and Z² are each H.

Also included in the invention are compounds represented by thefollowing Formula (II-79): ##STR11## wherein R¹ is CH₂ S(CH₂)₂ NH n--C₄H₉, X is CO₂ CH₃ ; R is OH; and R², Z¹, and Z² are each H.

Also included in the invention are compounds represented by thefollowing Formula (II-80): ##STR12## wherein R¹ is CH₂ S(CH₂)₂ N(CH₃)₂ ;R² is CH₂ S(CH₂)₂ N(CH₃)₂ ; X is CO₂ CH₃ ; R is OH; and Z¹ and Z² areeach H.

Also included in the invention are compounds represented by thefollowing Formula (V): ##STR13## in which X represents CO₂ R⁵ (in whichR⁵ represents lower alkyl) or CH₂ NHCO₂ R⁶ (in which R⁶ represents loweralkyl or aryl); R¹ represents hydrogen or CH₂ SO₂ R⁷ (in which R⁷represents lower alkyl), provided that the combination of X=CO₂ R⁵ andR¹ =hydrogen is excluded.

In the definitions of the groups in Formula (V), lower alkyl means astraight-chain or branched alkyl group having 1 to 6 carbon atoms,preferably 1 to 3 carbon atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl,and hexyl. Aryl means an aryl group having 6 to 10 carbon atoms, such asphenyl and naphthyl.

Also included in the invention are compounds (VI-1) represented by thefollowing Formula (VI): ##STR14## wherein X is CO₂ CH₃ ; R is OH; eachR¹, R², Z¹, and Z² is H; and R⁸ is NHCONHC₂ H₅.

Also included in the invention are compounds (VI-2) represented by theFormula (VI): ##STR15## wherein X is CO₂ CH₃ ; each R² and R⁸ is NH₂ ; Ris OH; and each R¹, Z¹, and Z² is H.

The compounds of the invention can be in the form of pharmaceuticallyacceptable salts including pharmaceutically acceptable acid additionsalts, metal salts, ammonium salts, organic amine addition salts, andamino acid addition salts.

Examples of the pharmaceutically acceptable acid addition salts areinorganic acid addition salts such as hydrochloride, sulfate, andphosphate; and organic acid addition salts such as acetate, maleate,fumarate, tartrate, and citrate. Examples of the pharmaceuticallyacceptable metal salts are alkali metal salts such as sodium salt andpotassium salt, alkaline earth metal salts such as magnesium salt andcalcium salt, aluminium salt, and zinc salt. Examples of thepharmaceutically acceptable ammonium salts are ammonium salt andtetraethyl ammonium salt. Examples of the pharmaceutically acceptableorganic amine addition salts are salts with morpholine and piperidine.Examples of the pharmaceutically acceptable amino acid addition saltsare salts with lysine, glycine, and phenylalanine.

In another aspect, the invention features a method for enhancing thefunction of cholinergic neurons, striatal neurons, basal forebrainneurons, and sensory neurons, e.g., dorsal root ganglion neurons, byadministering to a mammal, e.g., a human, a therapeutic amount of one ofthe novel bis-substituted derivatives of staurosporine. The therapy maybe given in conjunction with a trophic factor, preferably a member ofthe neurotrophin family, and most preferably nerve growth factor (NGF).As used herein, a "trophic factor" is a molecule that directly orindirectly affects the survival or function of a trophicfactor-responsive cell. The neurotrophin family is a group of proteinswith significant homology to NGF and include, in addition to NGF,brain-derived neurotrophic factor (BDNF; Leibrock et al., Nature341:149-152, 1989); neutrotrophin-3 (NT-3; Hohn et al., Nature344:339-341, 1990); and neurotrophin-5 (NT-4/5; Berkemeier et al.,Neuron 7:857-866, 1991).

In another aspect, the invention features a method for protecting nervecells of a mammal, e.g., a human, from degeneration induced byexcitatory amino acids, by administering to the mammal a therapeuticamount of one of the novel bis-substituted derivatives of staurosporine.Conditions in which such degeneration may occur include Alzheimer'sdisease; motor neuron disease, e.g., amyotrophic lateral sclerosis;Parkinson's disease; cerebrovascular disease, e.g., ischemic conditions;AIDS dementia; epilepsy; Huntington's disease; and concussive orpenetrating injuries to the brain or spinal cord. The therapy may begiven in conjunction with a neurotrophic factor, preferably a member ofthe neurotrophin family, most preferably nerve growth factor (NGF).

In another aspect, the invention features a method for enhancing thefunction of cholinergic neurons, striatal neurons, basal forebrainneurons, and/or sensory neurons, e.g., dorsal root ganglion neurons, ina mammal, e.g., a human, by administering to the mammal a therapeuticamount of a functional derivative of K-252a, represented by theformulas: ##STR16## with any of the substitutions shown in Table 1,below. Preferably, the method for enhancing the function and/or survivalof a cholinergic neuron, striatal neuron, basal forebrain neuron, and/orsensory neuron, e.g., a dorsal root ganglion neuron, in a mammalinvolves administering an effective amount of, e.g., Compound II-3,II-20, II-30, II-33, II-38, II-49, II-51, II-65, II-69, II-72, II-73,II-79, II-80, VI-1, or VI-2 of Table 1 to the mammal. More preferably,the method for enhancing the function and/or survival of a cholinergicneuron, striatal neuron, basal forebrain neuron, or sensory neuon in amammal involves administering an effective amount of Compound II-51.

                                      TABLE 1                                     __________________________________________________________________________                                                  Z.sup.1(1)                      Compound                                                                            R.sup.1      R.sup.2    X           R   Z.sup.2                         __________________________________________________________________________    II-1  H            H          CH.sub.2 N.sub.3                                                                          OH  H                               II-2  NHCONHC.sub.6 H.sub.5                                                                      H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-3  CH.sub.2 SOC.sub.2 H.sub.5                                                                 H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-4  H            H          CH.sub.2 OH OCH.sub.3                                                                         H                               II-5  H            H          CONHC.sub.2 H.sub.5                                                                       OH  H                               II-6  H            H                                                                                         ##STR17##  OH  H                               II-7.sup.(2,7)                                                                      H            H          CH.sub.2 NHGly                                                                            OH  H                               II-8  H            H          CON(CH.sub.3).sub.2                                                                       OH  H                               II-9.sup.(3)                                                                        H            H          CH.sub.2 NHCO.sub.2                                                                           H                               II-10 Br           H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-11 H            H          CONH.sub.2  OH  H                               II-12 H            H          CH.sub.2 OH OH  H                               II-13 H            H          CONHC.sub.3 H.sub.7                                                                       OH  H                               II-14.sup.(2)                                                                       H            H          CH.sub.2 NHSer                                                                            OH  H                               II-15 H            H          CH.sub.2 SOCH.sub.3                                                                       OH  H                               II-16 H            H          CHNOH       OH  H                               II-17 H            H                                                                                         ##STR18##  OH  H                               II-18.sup.(2,7)                                                                     H            H          CH.sub.2 NHPro                                                                            OH  H                               II-19 H            H          CHNNHC(NH)NH.sub.2                                                                        OH  H                               II-20 Br           Br         CO.sub.2 CH.sub.3                                                                         OH  O                               II-21 H            H          CONH(CH.sub.2).sub.2 OH                                                                   OH  H                               II-22 H            H          CO.sub.2 CH.sub.3                                                                         OH  O                               II-23 H            H          H           OH  H                               II-24 H            H          CHNNHCONH.sub.2                                                                           OH  H                               II-25 H            H          CH.sub.2 OCOCH.sub.3                                                                      OH  H                               II-26.sup.(3)                                                                       H            H          CH.sub.2 OC(CH.sub.3).sub.2 O                                                                 H                               II-29 NHCONHC.sub.2 H.sub.5                                                                      H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-30 CH.sub.2 SC.sub.2 H.sub.5                                                                  H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-31 Br           H          CH.sub.2 OH OH  H                               II-32 Br           Br         CO.sub.2 CH.sub.3                                                                         OH  H                               II-33 CH.sub.2 SC.sub.6 H.sub.5                                                                  H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-34 Cl           Cl         CO.sub.2 CH.sub.3                                                                         OH  H                               II-36 H            H          CONHC.sub.6 H.sub.5                                                                       OH  H                               II-37 H            H                                                                                         ##STR19##  OH  H                               II-38 H            H          CH.sub.2 NHCO.sub.2 C.sub.6 H.sub.5                                                       OH  H                               II-39 NHCONHC.sub.2 H.sub.5                                                                      NHCONHC.sub.2 H.sub.5                                                                    CO.sub.2 CH.sub.3                                                                         OH  H                               II-40 N(CH.sub.3).sub.2                                                                          H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-41 CH.sub.3     H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-42 CH.sub.2 OCONHC.sub.2 H.sub.5                                                              H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-43 NHCO.sub.2 CH.sub.3                                                                        H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-44 Br           Br         CH.sub.2 OH OH  H                               II-45 Br           Br         CONHC.sub.6 H.sub.5                                                                       OH  H                               II-46 Br           Br         CONHCH.sub.2 CH.sub.2 OH                                                                  OH  H                               II-47 CH.sub.2 OC.sub.2 H.sub.5                                                                  H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-48 CH.sub.2 N(CH.sub.3).sub.2                                                                 H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-49 CH.sub.2 SO.sub.2 C.sub.2 H.sub.5                                                          H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-50                                                                                ##STR20##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-51 CH.sub.2 SC.sub.2 H.sub.5                                                                  CH.sub.2 SC.sub.2 H.sub.5                                                                CO.sub.2 CH.sub.3                                                                         OH  H                               II-52                                                                                ##STR21##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-53                                                                                ##STR22##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-54                                                                                ##STR23##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-55                                                                                ##STR24##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-56 CH.sub.2 SC.sub.2 H.sub.5                                                                  CH.sub.2 OH                                                                              CO.sub.2 CH.sub.3                                                                         OH  H                               II-57 H            H          CH.sub.2 NHCO.sub.2 CH.sub.3                                                              OH  H                               II-58 Br           H          CONH.sub.2  OH  H                               II-59 H            H          CH.sub.2 SC.sub.6 H.sub.5                                                                 OH  H                               II-60 H            H                                                                                         ##STR25##  OH  H                               II-61 H            H          CH.sub.2 SOC.sub.6 H.sub.5                                                                OH  H                               II-62 H            H          CO.sub.2 n-hexyl                                                                          OH  H                               II-63 OH           H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-64 O n-propyl   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-65 CH.sub.2 SCH.sub.2 CH.sub.2 N(CH.sub.3).sub.2                                              H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-66 H            H          CH.sub.2 NH.sub.2                                                                         OH  H                               II-67 H            H          CONHCH.sub.3                                                                              OH  H                               II-68                                                                                ##STR26##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-69                                                                                ##STR27##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-70                                                                                ##STR28##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-71                                                                                ##STR29##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-72 CH.sub.2 S(CH.sub.2).sub.2 NH.sub.2                                                        H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-73                                                                                ##STR30##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-74 CHNNHC(NH)NH.sub.2                                                                         H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-75                                                                                ##STR31##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-76                                                                                ##STR32##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-77 CHNN(CH.sub.3).sub.2                                                                       H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-78                                                                                ##STR33##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-79 CH.sub.2 S(CH.sub.2).sub.2 NHn-C.sub.4 H.sub.9                                             H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-80 CH.sub.2 S(CH.sub.2).sub.2 N(CH.sub.3).sub.2                                               CH.sub.2 S(CH.sub.2).sub.2N(CH.sub.3).sub.2                                              CO.sub.2 CH.sub.3                                                                         OH  H                               II-81 CH.sub.2 SCH(CH.sub.3).sub.2                                                               CH.sub.2 SCH(CH.sub.3).sub.2                                                             CO.sub.2 CH.sub.3                                                                         OH  H                               II-82 CH.sub.2 S(CH.sub.2).sub.2 CH.sub.3                                                        CH.sub.2 S(CH.sub.2).sub.2 CH.sub.3                                                      CO.sub.2 CH.sub.3                                                                         OH  H                               II-83 CH.sub.2 S(CH.sub.2).sub.3 CH.sub.3                                                        CH.sub.2 S(CH.sub.2).sub.3 CH.sub.3                                                      CO.sub.2 CH.sub.3                                                                         OH  H                               II-84 CH.sub.2 OCH.sub.3                                                                         CH.sub.2 OCH.sub.3                                                                       CO.sub.2 CH.sub.3                                                                         OH  H                               II-85 CH.sub.2 OC.sub.2 H.sub.5                                                                  CH.sub.2 OC.sub.2 H.sub.5                                                                CO.sub.2 CH.sub.3                                                                         OH  H                               II-86 CH.sub.2 OH  NHCONHC.sub.2 H.sub.5                                                                    CO.sub.2 CH.sub.3                                                                         OH  H                               II-87 CH.sub.2 SC.sub.2 H.sub.5                                                                  NHCONHC.sub.2 H.sub.5                                                                    CO.sub.2 CH.sub.3                                                                         OH  H                               II-88 CH.sub.3     CH.sub.3   CO.sub.2 CH.sub.3                                                                         OH  H                               II-89 CH.sub.2 SC.sub.2 H.sub.5                                                                  CH.sub.2 S(O)C.sub.2 H.sub.5                                                             CO.sub.2 CH.sub.3                                                                         OH  H                               II-90 CH.sub.2 OH  CH.sub.2 OH                                                                              CO.sub.2 CH OH  H                               II-91 CH(SCH.sub.2 CH.sub.2 S)                                                                   NO.sub.2   CO.sub.2 CH.sub.3                                                                         OH  H                               II-92 CH(SCH.sub.2 CH.sub.2 S)                                                                   NHCONHC.sub.2 H.sub.5                                                                    CO.sub.2 CH.sub.3                                                                         OH  H                               III-1 --           --         --          --  H                               III-2 --           --         --          --  O                               IV-1.sup.(4,9)                                                                      H            H          --          --  H                               IV-2.sup.(5)                                                                        Br           H          --          --  H                               IV-3.sup.(6)                                                                        H            H          --          --  H                               IV-4.sup.(8,9)                                                                      H            H          --          --  H                               IV-5.sup.(10)                                                                       H            H          --          --  H                               IV-6.sup.(7,11)                                                                     H            H          --          --  H                               VI-1.sup.(12)                                                                       H            H          CO.sub.2 CH.sub.3                                                                         OH  H                               VI-2.sup.(13)                                                                       H            NH.sub.2   CO.sub.2 CH.sub.3                                                                         OH  H                               __________________________________________________________________________     .sup.(1) Z.sup.1 and Z.sup.2 are both hydrogen, or both are combined          together to represent oxygen, where indicated.                                .sup.(2) NHamino acid linkage is an amide bond through the carboxyl group     of the amino acid.                                                            .sup.(3) X and R are combined together to form the linking group.             .sup.(4) R.sup.3 is CH.sub.2 CHCH.sub.2 ; R.sup.4 is H.                       .sup.(5) R.sup.3 and R.sup.4 are each H.                                      .sup.(6) R.sup.3 and R.sup.4 are each CH.sub.2 CHCH.sub.2.                    .sup.(7) Compound is in the form of the hydrochloride.                        .sup.(8) R.sup.3 is H and R.sup.4 is CH.sub.2 CHCH.sub.2.                     .sup.(9) IV1 and IV4 are a 1.5 to 1.0 mixture of the two components.          .sup.(10) R.sup.3R.sup.4CH.sub.2 CH.sub.2 CH.sub.2 OH                         .sup.(11)                                                                     ##STR34##                                                                     .sup.(12) R.sup.8NHCONHC.sub.2 H.sub.5.                                       .sup.(13) R.sup.8NH.sub.2.                                               

The therapy may be given in conjunction with a trophic factor,preferably a member of the neurotrophin family, most preferably nervegrowth factor (NGF).

In a preferred aspect, the invention features a method for enhancing thefunction of a dorsal root ganglion nerve cell, by administering to amammal, e.g., a human, a therapeutic amount of a functional derivativeof K-252a, represented by the formula (II) or (III): ##STR35## whereinthe following substitutions are made:

                                      TABLE 2                                     __________________________________________________________________________                                     Z.sup.1(2)                                   Compound.sup.(1)                                                                     R.sup.1  X            R   Z.sup.2                                      __________________________________________________________________________    II-1   H        CH.sub.2 N.sub.3                                                                           OH  H                                            II-2   NHCONHC.sub.6 H.sub.5                                                                  CO.sub.2 CH.sub.3                                                                          OH  H                                            II-3   CH.sub.2 SOC.sub.2 H.sub.5                                                             CO.sub.2 CH.sub.3                                                                          OH  H                                            II-4   H        CH.sub.2 OH  OCH.sub.3                                                                         H                                            II-5   H        CONHC.sub.2 H.sub.5                                                                        OH  H                                            II-6   H                                                                                       ##STR36##   OH  H                                            II-8   H        CON(CH.sub.3).sub.2                                                                        OH  H                                            II-9.sup.(3)                                                                         H        CH.sub.2 NHCO.sub.2                                                                            H                                            II-10  Br       CO.sub.2 CH.sub.3                                                                          OH  H                                            II-11  H        CONH.sub.2   OH  H                                            II-12  H        CH.sub.2 OH  OH  H                                            III-1  --       --           --  H                                            II-13  H        CONHC.sub.3 H.sub.7                                                                        OH  H                                            II-15  H        CH.sub.2 SOCH.sub.3                                                                        OH  H                                            II-17  H                                                                                       ##STR37##   OH  H                                            II-19  H        CHNNHC(NH)NH.sub.2                                                                         OH  H                                            II-20.sup.(1)                                                                        Br       CO.sub.2 CH.sub.3                                                                          OH  O                                            II-21  H        CONH(CH.sub.2).sub.2 OH                                                                    OH  H                                            III-2  --       --           --  O                                            II-23  H        H            OH  H                                            II-24  H        CHNNHCONH.sub.2                                                                            OH  H                                            II-25  H        CH.sub.2 OCOCH.sub.3                                                                       OH  H                                            II-30  CH.sub.2 SC.sub.2 H.sub.5                                                              CO.sub.2 CH.sub.3                                                                          OH  H                                            II-32  Br       CO.sub.2 CH.sub.3                                                                          OH  H                                            __________________________________________________________________________     .sup.(1) R.sup.2 is hydrogen, except in compound II20 and II32 where          R.sup.2Br.                                                                    .sup.(2) Z.sup.1 and Z.sup.2 are both hydrogen, or both are combined          together to represent oxygen, where indicated.                                .sup.(3) X and R are combined together to form the linking group.        

The therapy may be given in conjunction with a neurotrophic factor,preferably a member of the neurotrophin family, most preferably nervegrowth factor (NGF).

In a preferred aspect, the invention features a method for enhancing thefunction of cholinergic neurons of a mammal, e.g., a human, byadministering to the mammal a therapeutic amount of K-252a, representedby the formula (II): ##STR38## wherein R¹ and R² are each H, X is CO₂CH₃, R is OH, and Z¹ and Z² are each H. The therapy may be given inconjunction with a trophic factor, preferably a member of theneurotrophin family, most preferably nerve growth factor (NGF).

In a preferred aspect, the invention features a method for enhancing thesurvival and/or function of a striatal nerve cell, by administering to amammal, e.g., a human, a therapeutic amount of K-252a or a functionalderivative of K-252a, represented by the formulae (II), (III), or (IV):##STR39## wherein the following substitutions are made:

                  TABLE 3                                                         ______________________________________                                        Com-                                      Z.sup.2(1)                          pound R.sup.1     R.sup.2                                                                             X            R    Z.sup.1                             ______________________________________                                        K-252a                                                                              H           H     CO.sub.2 CH.sub.3                                                                          OH   H                                   III-1 --          --    --           --   H                                   II-1  H           H     CH.sub.2 N   OH   H                                   II-35 H           H     CO.sub.2 n-C.sub.6 H.sub.13                                                                OH   H                                   II-20 Br          Br    CO.sub.2 CH.sub.3                                                                          OH   O                                   II-10 Br          H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-28 O-n-C.sub.3 H.sub.7                                                                       H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-5  H           H     CONHC.sub.2 H.sub.5                                                                        OH   H                                   II-29 NHCONHC.sub.2 H.sub.5                                                                     H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-2  NHCONHC.sub.6 H.sub.5                                                                     H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-3  CH.sub.2 SOC.sub.2 H.sub.5                                                                H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-30 CH.sub.2 SC.sub.2 H.sub.5                                                                 H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-6  H           H                                                                                    ##STR40##   OH   H                                   II-31 Br          H     CH.sub.2 OH  OH   H                                   II-32 Br          Br    CO.sub.2 CH.sub.3                                                                          OH   H                                   IV-1.sup.(2)                                                                        --          H     --           --   H                                   II-33 CH.sub.2 SC.sub.6 H.sub.5                                                                 H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-34 Cl          Cl    CO.sub.2 CH.sub.3                                                                          OH   H                                   ______________________________________                                         .sup.(1) Z.sup.1 and Z.sup.2 are both hydrogen, or both are combined          together to represent oxygen, where indicated.                                .sup.(2) R.sup.3 is CH.sub.2CHCH.sub.2 ; R.sup.4 is H.                   

In another aspect, the invention features a method for enhancing thesurvival and/or function of a basal forebrain nerve cell, byadministering to a mammal, e.g., a human, a therapeutic amount of K-252aor a functional derivative of K-252a, represented by the formula (II):##STR41## wherein the following substitutions are made:

                                      TABLE 4                                     __________________________________________________________________________    Compound                                                                             R.sup.1     R.sup.2                                                                              X        R   Z.sup.2, Z.sup.1(1)                    __________________________________________________________________________    K-252a H           H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-3   CH.sub.2 SOC.sub.2 H.sub.5                                                                H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-5   H           H      CONHC.sub.2 H.sub.5                                                                    OH  H                                      II-10  Br          H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-20  Br          Br     CO.sub.2 CH.sub.3                                                                      OH  O                                      II-21  H           H      CONH(CH.sub.2).sub.2 OH                                                                OH  H                                      II-22  H           H      CO.sub.2 CH.sub.3                                                                      OH  O                                      II-30  CH.sub.2 SC.sub.2 H.sub.5                                                                 H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-32  Br          Br     CO.sub.2 CH.sub.3                                                                      OH  H                                      II-51  CH.sub.2 SC.sub.2 H.sub.5                                                                 CH.sub.2 SC.sub.2 H.sub.5                                                            CO.sub.2 CH.sub.3                                                                      OH  H                                      II-62  H           H      CO.sub.2 n-hexyl                                                                       OH  H                                      II-63  OH          H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-64  O n-propyl  H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-65  CH.sub.2 SCH.sub.2 CH.sub.2 N(CH.sub.3).sub.2                                             H      CO.sub.2 CH.sub.3                                                                      OH  H                                      __________________________________________________________________________     .sup.(1) Z.sup.1 and Z.sup.2 are both hydrogen, or both are combined          together to represent oxygen, where indicated.                           

The therapy may be given in conjunction with a trophic factor,preferably a member of the neurotrophin family, most preferably nervegrowth factor.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS THE DRAWINGS ARE FIRSTDESCRIBED.

Drawings

FIG. 1 is a graph illustrating the effect of the K-252a derivatives1,6-hexamethylene-bis-(carbamylstaurosporine) (HBCS) and staurosporineon basal ornithine decarboxylase (ODC) activity in PC-12 cells.

FIG. 2 is a graph illustrating the effects of staurosporine, HBCS, andK-252a on NGF-stimulated ODC activity in PC-12 cells.

FIG. 3 is a graph illustrating the NGF-potentiating effect of HBCS onODC activity in PC-12 cells.

FIG. 4 is a graph illustrating the effect of K-252a on cholineacetyltransferase (CHAT) specific activity in rat embryonic spinal cordcultures.

FIG. 5 is a graph illustrating the time course of K-252a effect on ChATactivity in rat embryonic spinal cord cultures.

FIG. 6 is a graph illustrating the effect of K-252a on survival of chickembryonic dorsal root ganglion neurons.

FIG. 7 is a graph illustrating the effect of K-252a functionalderivatives on survival of chick embryonic dorsal root ganglion neurons.

FIG. 8 is a graph illustrating the effect of K-252a functionalderivatives on ChAT activity in rat embryonic spinal cord cultures.

FIG. 9 is a graph illustrating the effect of K-252a on kainate-induceddamage to the rat hippocampus.

FIG. 10 is a graph illustrating the effect of K-252a on kainate-inducedspectrin proteolysis in the rat hippocampus.

FIG. 11 is a graph illustrating the effect of HBCS on kainate-induceddamage to the hippocampus.

FIG. 12 is a graph illustrating the effect of K-252a functionalderivatives on kainate-induced spectrin proteolysis in the rathippocampus.

FIGS. 13a, 13b and 13c are tables showing the relative activity ofK-252a derivatives on ChAT activity in rat spinal cord cultures.

FIG. 14 is a table showing the relative activity of K-252a derivativeson neuronal survival in chick dorsal root ganglion cultures.

FIG. 15 is a graph illustrating survival of striatal neurons in thepresence of K-252a.

FIG. 16 is a graph illustrating the time course of survival of striatalcells in the presence of K-252a.

FIG. 17 is a pair of photomicrographs of striatal neurons cultured inthe presence or absence of K-252a.

FIG. 18 is a table showing the relative activity of K-252a derivativeson neuronal survival in rat striatal cultures.

FIG. 19 is a table showing the relative activity of K-252a derivativeson the survival of low density basal forebrain neurons.

FIG. 20 is a bar graph demonstrating that Compound II-51 preventsdevelopmental programmed motoneuron death in ovo.

FIG. 21 is a photographic demonstration that Compound II-51 prevents theaxotomy-induced loss of CHAT immunoreactivity in the adult hypoglossalnucleus.

FIG. 22 is a diagram showing the synthesis of Compound H from startingCompound C.

FIG. 23 is a diagram showing the synthesis of Compound II-45 fromstarting Compound J.

FIG. 24 is a diagram showing the structure of Compound P, Compound Q,and Compound R.

FIG. 25 is a diagram showing the synthesis of Compound IV-6 fromstarting Compound S.

FIG. 26 is a diagram showing the chemical structure of compounds (AA),(BB), (CC), (DD) and (EE).

FIG. 27 is a diagram showing the chemical structure of compounds (FF),(GG), (HH) and (JJ).

STAUROSPORINE DERIVATIVES

The present invention relates to novel bis-N-substituted derivatives ofstaurosporine and their use as therapeutics for neurological diseases,especially those diseases characterized either by neuronal cells whichare injured, compromised, undergoing axonal degeneration, or atincreased risk of dying, or by impaired cholinergic activity. Thesediseases include those induced by excitatory amino acids. Thetherapeutic use of these novel derivatives includes use of thederivatives alone and use of the derivatives combined with exogenousadministration of neurotrophic factors (preferably members of theneurotrophin family, most preferably NGF. The compounds within the scopeof this invention may be represented by the formula

     Stau!-N(CH.sub.3)--W--N(CH.sub.3)- Stau!                  (I)

in which Stau! represents a residue of the formula: ##STR42## and Wrepresents a bis(carbamyl) or bis(thiocarbamyl) radical,

    --C(═Y)--NH--W'--NH--C(═Y)--

in which W' is a hydrocarbylene radical of 2-20 carbon atoms and Y is Oor S. W' is preferably an alkylene radical of 2-10 carbons,unsubstituted, or substituted with 1-3 alkyl groups of 1-3 carbons; anarylene radical of 6-12 carbons, unsubstituted, or substituted with 1-3alkyl groups of 1-3 carbons, chlorine or bromine. W' is especiallypreferably hexamethylene and 1,4-phenylene. Y is preferably O.

Compounds of formula (I) can be prepared by procedures known in the artfor preparation of carbamates and thiocarbamates. Preferably, thecompounds are prepared by reaction of a bis-diisocyanate or abis-diisothiocyanate with staurosporine to give a compound of formula(I) wherein Y=O or Y=S respectively.

Intermediate bis-diisocyanates and bis-diisothiocyanates suitable foruse include:

1,6-diisocyanatohexane

toluene-2,6-diisocyanate

benzene-1,2-diisocyanate

2-methyl-1,5-diisocyanatopentane

naphthalene-2,6 diisocyanate

1,6-diisothiocyanatohexane

1,4-diisothiocyanatobutane

toluene-2,4-diisocyanate

benzene-1,4-diisocyanate

1,2-diisocyanatoethane

naphthalene-1,5-diisocyanate

1,5-diisocyanatopentane

benzene-1,4-diisothiocyanate

2-methyl-1,5-diisothiocyanatopentane

For reviews of the preparation of isocyanates and isothiocyanates, seeRichter and Ulrich, pp. 619-818, in The Chemistry of Cyanates and TheirThio Derivatives, Part 2, (Patai, ed.) Wiley, New York, 1977. Thecompounds are preferably prepared by reaction of phosgene (Y=O) orthiophosgene (Y=S) with the corresponding diamine. Alternative methodsof preparation may also be employed. For example, 1,2-diisocyanatoethanemay be prepared by reaction of ethylene urea with phosgene followed byheating.

K-252a Derivatives

The present invention is also directed to the use of specific functionalderivatives of K-252a, as therapeutics in certain neurological diseasesor disturbances characterized by neurons which are injured, compromised,undergoing axonal degeneration, or at risk of dying. The functionalderivatives may be administered alone or in conjunction with aneurotrophic factor (preferably a member of the neurotrophin family,most preferably nerve growth factor, NGF).

A "functional derivative" of K-252a is defined as a modified form ofthat molecule, which possesses the desired biological activity, hereindefined as neuroprotective activity, for example the ability to promotenerve cell survival, or to promote nerve fiber (e.g. axonal) growth, orto enhance cholinergic nerve cell function, or to enhance the functionof sensory cells, e.g., dorsal root ganglion nerve cells, or to enhancethe function and/or survival of striatal neurons, or to enhance thefunction and/or survival of basal forebrain neurons. Such molecularmodifications may improve the molecule's solubility, absorption,transport (e.g., through the blood-brain barrier and cellularmembranes), biological halflife, etc. Alternatively, or in addition,some moieties may decrease the toxicity of the molecule, or eliminate orattenuate any undesirable side effect of the molecule.

The compounds within the scope of the invention may be represented byformula (II) hereinafter referred to as compound (II)!, formula (III)hereinafter referred to as compound (III)!, formula (IV) hereinafterreferred to as compound (IV)!, formula (V) hereinafter referred to ascompound (V)!, and formula (VI) hereinafter referred to as compound(VI)!, below: ##STR43## with substitutions in Table 5, below. Thefunctional derivatives of K-252a of the invention may be prepared denovo by chemical synthesis using methods known to those skilled in theart. For example, procedures used for preparation of Compound II aredescribed by Murakata et al (U.S. Pat. No. 4,923,986), herebyincorporated by reference. Procedures used for preparation of CompoundIII are described by Moody et al., J. Org. Chem. 57: 2105-2114 (1992);Steglich et al., Angew. Chem. Int. Ed. Engl. 19: 459-460 (1980);Nakanishi et al., J. Antibiotics 39: 1066-1071 (1986); and JapanesePatent Application No. 60-295172 (1985). Further methods are describedfor compounds II-1, 9, 12 and 15 in Japanese Patent Application No.60-295173 (1985); compounds II-2, 3, 4, 24, 25 and 26 in Japanese PatentApplication No. 62-327858 (1987); compounds II-20 in Japanese PatentApplication No. 62-327859 (1987); and compounds II-10 in Japanese PatentApplication No. 60-257652 (1985) by Meiji Seika Kaisha Ltd.

                                      TABLE 5                                     __________________________________________________________________________    Functional Derivatives of K-252a.sup.(12)                                                                                   Z.sup.1(1)                      Compound                                                                            R.sup.1      R.sup.2    X           R   Z.sup.2                         __________________________________________________________________________    II-1  H            H          CH.sub.2 N.sub.3                                                                          OH  H                               II-2  NHCONHC.sub.6 H.sub.5                                                                      H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-3  CH.sub.2 SOC.sub.2 H.sub.5                                                                 H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-4  H            H          CH.sub.2 OH OCH.sub.3                                                                         H                               II-5  H            H          CONHC.sub.2 H.sub.5                                                                       OH  H                               II-6  H            H                                                                                         ##STR44##  OH  H                               II-7.sup.(2,7)                                                                      H            H          CH.sub.2 NHGly                                                                            OH  H                               II-8  H            H          CON(CH.sub.3).sub.2                                                                       OH  H                               II-9.sup.(3)                                                                        H            H          CH.sub.2 NHCO.sub.2                                                                           H                               II-10 Br           H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-11 H            H          CONH.sub.2  OH  H                               II-12 H            H          CH.sub.2 OH OH  H                               II-13 H            H          CONHC.sub.3 H.sub.7                                                                       OH  H                               II-14.sup.(2)                                                                       H            H          CH.sub.2 NHSer                                                                            OH  H                               II-15 H            H          CH.sub.2 SOCH.sub.3                                                                       OH  H                               II-16 H            H          CHNOH       OH  H                               II-17 H            H                                                                                         ##STR45##  OH  H                               II-18.sup.(2,7)                                                                     H            H          CH.sub.2 NHPro                                                                            OH  H                               II-19 H            H          CHNNHC(NH)NH.sub.2                                                                        OH  H                               II-20 Br           Br         CO.sub.2 CH.sub.3                                                                         OH  O                               II-21 H            H          CONH(CH.sub.2).sub.2 OH                                                                       OHH                             II-22 H            H          CO.sub.2 CH.sub.3                                                                         OH  O                               II-23 H            H          H           OH  H                               II-24 H            H          CHNNHCONH.sub.2 OHH                             II-25 H            H          CH.sub.2 OCOCH.sub.3                                                                      OH  H                               II-26.sup.(3)                                                                       H            H          CH.sub.2 OC(CH.sub.3).sub.2 O                                                                 H                               II-29 NHCONHC.sub.2 H.sub.5                                                                      H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-30 CH.sub.2 SC.sub.2 H.sub.5                                                                  H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-31 Br           H          CH.sub.2 OH OH  H                               II-32 Br           Br         CO.sub.2 CH.sub.3                                                                         OH  H                               II-33 CH.sub.2 SC.sub.6 H.sub.5                                                                  H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-34 Cl           Cl         CO.sub.2 CH.sub.3                                                                         OH  H                               II-36 H            H          CONHC.sub.6 H.sub.5                                                                       OH  H                               II-37 H            H                                                                                         ##STR46##  OH  H                               II-38 H            H          CH.sub.2 NHCO.sub.2 C.sub.6 H.sub.5                                                           OH H                            II-39 NHCONHC.sub.2 H.sub.5                                                                      NHCONHC.sub.2 H.sub.5                                                                    CO.sub.2 CH.sub.3                                                                         OH  H                               II-40 N(CH.sub.3).sub.2                                                                          H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-41 CH.sub.3     H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-42 CH.sub.2 OCONHC.sub.2 H.sub.5                                                              H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-43 NHCO.sub.2 CH.sub.3                                                                        H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-44 Br           Br         CH.sub.2 OH OH  H                               II-45 Br           Br         CONHC.sub.6 H.sub.5                                                                       OH  H                               II-46 Br           Br         CONHCH.sub.2 CH.sub.2 OH                                                                      OH H                            II-47 CH.sub.2 OC.sub.2 H.sub.5                                                                  H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-48 CH.sub.2 N(CH.sub.3).sub.2                                                                 H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-49 CH.sub.2 SO.sub.2 C.sub.2 H.sub.5                                                          H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-50                                                                                ##STR47##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-51 CH.sub.2 SC.sub.2 H.sub.5                                                                  CH.sub.2 SC.sub.2 H.sub.5                                                                CO.sub.2 CH.sub.3                                                                         OH  H                               II-52                                                                                ##STR48##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-53                                                                                ##STR49##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-54                                                                                ##STR50##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-55                                                                                ##STR51##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-56 CH.sub.2 SC.sub.2 H.sub.5                                                                  CH.sub.2 OH                                                                              CO.sub.2 CH.sub.3                                                                         OH  H                               II-57 H            H          CH.sub.2 NHCO.sub.2 CH.sub.3                                                              OH  H                               II-58 Br           H          CONH.sub.2  OH  H                               II-59 H            H          CH.sub.2 SC.sub.6 H.sub.5                                                                 OH  H                               II-60 H            H                                                                                         ##STR52##  OH  H                               II-61 H            H          CH.sub.2 SOC.sub.6 H.sub.5                                                                OH  H                               II-62 H            H          CO.sub.2 n-hexyl                                                                          OH  H                               II-63 OH           H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-64 O n-propyl   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-65 CH.sub.2 SCH.sub.2 CH.sub.2 N(CH.sub.3).sub.2                                              H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-66 H            H          CH.sub.2 NH.sub.2                                                                         OH  H                               II-67 H            H          CONHCH.sub.3                                                                              OH  H                               II-68                                                                                ##STR53##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-69                                                                                ##STR54##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-70                                                                                ##STR55##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-71                                                                                ##STR56##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-72 CH.sub.2 S(CH.sub.2).sub.2 NH.sub.2                                                        H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-73                                                                                ##STR57##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-74 CHNNHC(NH)NH.sub.2                                                                         H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-75                                                                                ##STR58##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-76                                                                                ##STR59##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-77 CHNN(CH.sub.3).sub.2                                                                       H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-78                                                                                ##STR60##   H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-79 CH.sub.2 S(CH.sub.2).sub.2 NH-n-C.sub.4 H.sub.9                                            H          CO.sub.2 CH.sub.3                                                                         OH  H                               II-80 CH.sub.2 S(CH.sub.2).sub.2 N(CH.sub.3).sub.2                                               CH.sub.2 S(CH.sub.2).sub.2N(CH.sub.3).sub.2                                              CO.sub.2 CH.sub.3                                                                         OH  H                               II-81 CH.sub.2 SCH(CH.sub.3).sub.2                                                               CH.sub.2 SCH(CH.sub.3).sub.2                                                             CO.sub.2 CH.sub.3                                                                         OH  H                               II-82 CH.sub.2 S(CH.sub.2).sub.2 CH.sub.3                                                        CH.sub.2 S(CH.sub.2).sub.2 CH.sub.3                                                      CO.sub.2 CH.sub.3                                                                         OH  H                               II-83 CH.sub.2 S(CH.sub.2).sub.3 CH.sub.3                                                        CH.sub.2 S(CH.sub.2).sub.3 CH.sub.3                                                      CO.sub.2 CH.sub.3                                                                         OH  H                               II-84 CH.sub.2 OCH.sub.3                                                                         CH.sub.2 OCH.sub.3                                                                       CO.sub.2 CH.sub.3                                                                         OH  H                               II-85 CH.sub.2 OC.sub.2 H.sub.5                                                                  CH.sub.2 OC.sub.2 H.sub.5                                                                CO.sub.2 CH.sub.3                                                                         OH  H                               II-86 CH.sub.2 OH  NHCONHC.sub.2 H.sub.5                                                                    CO.sub.2 CH.sub.3                                                                         OH  H                               II-87 CH.sub.2 SC.sub.2 H.sub.5                                                                  NHCONHC.sub.2 H.sub.5                                                                    CO.sub.2 CH.sub.3                                                                         OH  H                               II-88 CH.sub.3     CH.sub.3   CO.sub.2 CH.sub.3                                                                         OH  H                               II-89 CH.sub.2 SC.sub.2 H.sub.5                                                                  CH.sub.2 S(O)C.sub.2 H.sub.5                                                             CO.sub.2 CH.sub.3                                                                         OH  H                               II-90 CH.sub.2 OH  CH.sub.2 OH                                                                              CH.sub.2 OH OH  H                               II-91 CH(SCH.sub.2 CH.sub.2 S)                                                                   NO.sub.2   CO.sub.2 CH.sub.3                                                                         OH  H                               II-92 CH(SCH.sub.2 CH.sub.2 S)                                                                   NHCONHC.sub.2 H.sub.5                                                                    CO.sub.2 CH.sub.3                                                                         OH  H                               III-1 --           --         --          --  H                               III-2 --           --         --          --  O                               IV-1.sup.(4,9)                                                                      H            H          --          --  H                               IV-2.sup.(5)                                                                        Br           H          --          --  H                               IV-3.sup.(6)                                                                        H            H          --          --  H                               IV-4.sup.(8,9)                                                                      H            H          --          --  H                               IV-5.sup.(10)                                                                       H            H          --          --  H                               IV-6.sup.(7,11)                                                                     H            H          --          --  H                               VI-1.sup.(13)                                                                       H            H          CO.sub.2 CH.sub.3                                                                         OH  H                               VI-2.sup.(14)                                                                       H            NH.sub.2   CO.sub.2 CH.sub.3                                                                         OH  H                               __________________________________________________________________________     .sup.(1) Z.sup.1 and Z.sup.2 are both hydrogen, or both are combined          together to represent oxygen, where indicated.                                .sup.(2) NHamino acid linkage is an amide bond through the carboxyl group     of the amino acid.                                                            .sup.(3) X and R are combined together to form the linking group.             .sup.(4) R.sup.3 is CH.sub.2 CHCH.sub.2 ; R.sup.4 is H.                       .sup.(5) R.sup.3 and R.sup.4 are each H.                                      .sup.(6) R.sup.3 and R.sup.4 are each CH.sub.2 CHCH.sub.2.                    .sup.(7) Compound is in the form of the hydrochloride.                        .sup.(8) R.sup.3 is H and R.sup.4 is CH.sub.2 CHCH.sub.2.                     .sup.(9) IV1 and IV4 are a 1.5 to 1.0 mixture of the two components.          .sup.(10) R.sup.3R.sup.4CH.sub.2 CH.sub.2 CH.sub.2 OH                         .sup.(11)                                                                     ##STR61##                                                                     .sup.(12) For K252a itself, R.sup.1R.sup.2H, XCO.sub.2 CH.sub.3,              ROH, and Z.sup.1 and Z.sup.2 are each H.                                      (13) R.sup.8NHCONHC.sub.2 H.sub.5.                                            (14) R.sup.8NH.sub.2.                                                    

The invention also involves a method for enhancing the function ofcholinergic neurons, by administration of a therapeutic amount ofK-252a, represented by the formula (II) given above and substitutionsshown in Table 5 (note 12). This compound is prepared by proceduresdescribed in the art (see Matsuda et al., U.S. Pat. No. 4,554,402; Kaseet al., J. Antibiotics 37:1059-1065, 1986).

By "enhancing the function of cholinergic neurons" is meant promotingcholinergic nerve cell survival, and/or nerve fiber (e.g. axonal)growth, and/or enhancing cholinergic function of nerve cells. K-252a maybe administered with or without a trophic factor, preferably a member ofthe neurotrophin family, most preferably nerve growth factor (NGF).

Uses of the Compounds

As described more fully below, the present invention provides novel usesof functional derivatives of K-252a or compounds of Formula I, eitheralone or in combination with neurotrophic factors such as NGF, astherapeutics for neurological diseases, especially those diseasescharacterized either by neuronal cells which are injured, compromised,undergoing axonal degeneration, or at increased risk of dying, or byimpaired cholinergic activity. These diseases include those induced byexcitatory amino acids. The bioactivity of the compounds of theinvention, including the combination with a neurotrophic factor, mayconveniently be assayed by a cultured PC-12 cell ornithine decarboxylaseassay, a cultured spinal cord or basal forebrain cholineacetyltransferase assay, a cultured dorsal root ganglion neuron survivalassay, a cultured striatal neuron survival assay, a cultured basalforebrain neuron survival assay, an in ovo model of developmentallyprogrammed motoneuron death, an in vivo adult hypoglossal axotomy model,or an in vivo excitotoxin neuroprotection assay, e.g., a excitotoxiclesioning of the nucleus basalis. These assays are all described indetail below. Thus, the compounds of this invention are useful foradministration to humans or other mammals who suffer from neurologicaldiseases or disturbances characterized by increased risk of neuronalcell death or dysfunction. These neurological diseases and disturbancesinclude but are not limited to: Alzheimer's disease; motor neurondisease including amyotrophic lateral sclerosis; Parkinson's disease;stroke or other ischemic injuries; Huntington's disease; AIDS dementia;epilepsy; concussive or penetrating injuries of the brain or spinalcord; and peripheral neuropathies.

The compounds provided herein can be formulated into pharmaceuticalcompositions by admixture with pharmaceutically acceptable nontoxicexcipients and carriers. As noted above, such compositions may beprepared for use in parenteral administration, particularly in the formof liquid solutions or suspensions; for oral administration,particularly in the form of tablets or capsules; or intranasally,particularly in the form of powders, nasal drops, or aerosols.

The composition may conveniently be administered in unit dosage form andmay be prepared by any of the methods well known in the pharmaceuticalart, for example, as described in Remington's Pharmaceutical Sciences(Mack Pub. Co, Easton, Pa., 1980). Formulations for parenteraladministration may contain as common excipients sterile water or saline,polyalkylene glycols such as polyethylene glycol, oils of vegetableorigin, hydrogenated naphthalenes and the like. In particular,biocompatable, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be usefulexcipients to control the release of the active compounds. Otherpotentially useful parenteral delivery systems for these activecompounds include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation administration contain as excipients, for example, lactose,or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oilysolutions for administration in the form of nasal drops, or as a gel tobe applied intranasally. Formulations for parenteral administration mayalso include glycocholate for buccal administration, a salicylate forrectal administration, or citric acid for vaginal administration.Formulations for transdermal patches are preferrably lipophilicemulsions.

The materials of this invention can be employed as the sole active agentin a pharmaceutical or can be used in combination with other activeingredients, e.g., other growth factors which could facilitate neuronalsurvival or axonal growth in neurological diseases or disorders, forexample, peripheral neuropathy.

The concentrations of the compounds described herein in a therapeuticcomposition will vary depending upon a number of factors, including thedosage of the drug to be administered, the chemical characteristics(e.g., hydrophobicity) of the compounds employed, and the route ofadministration. In general terms, the compounds of this invention may beprovided in an aqueous physiological buffer solution containing about0.1 to 10% w/v compound for parenteral administration. Typical doseranges are from about 1 μg/kg to about 1 g/kg of body weight per day; apreferred dose range is from about 0.01 mg/kg to 100 mg/kg of bodyweight per day. The preferred dosage of drug to be administered islikely to depend on such variables as the type and extent of progressionof the neurological disease, the overall health status of the particularpatient, the relative biological efficacy of the compound selected, theformulation of the compound excipients, and its route of administration.

The present invention will be further illustrated by the followingexamples. These examples are not to be construed as limiting the scopeof the invention, which is to be determined solely by the appendedclaims.

EXAMPLE 1 1,6-Hexamethylene-bis-(carbamylstaurosporine) (HBCS)

A solution of 1.0 mg (2.15 micromoles) of staurosporine (KamiyaBiomedical Company, Thousand Oaks, Calif.) in 1.00 ml of ethyl acetate(dried over anhydrous magnesium sulfate) was treated with 17 microliters(1.08 micromoles) of a solution of 10.75 mg ofhexamethylene-bis-isocyanate in 1.0 ml of dried ethyl acetate. Thereaction mixture in an amber glass reaction vial was allowed to stand atroom temperature for two days. A crystalline deposit weighing 600micrograms was separated. Its composition was verified as HBCS by fastatom bombardment mass spectroscopy (FAB-MS). ##STR62##

This product and all of the subsequently described staurosporinederivatives were stored in non-actinic glass vials.

EXAMPLE 2 p-Phenylene-bis-(carbamylstaurosporine) (PBCS)

A solution of 1.0 mg of staurosporine (2.15 micromoles) in 1.00 ml ofdried ethyl acetate was treated with 45 microliters (1.08 micromoles) ofa solution prepared from 3.83 mg of p-phenylene diisocyanate (TransWorld Chemicals P1586-1) in 1.00 ml of dried ethyl acetate. The reactionmixture was allowed to stand overnight. A white precipitate deposited.Then 0.5 ml of petroleum ether was added. The mixture was filtered intoa vacuum-dried sintered-glass funnel. A total of 0.90 mg of crystallineproduct was collected and was identified asp-phenylene-bis-(carbamylstaurosporine) by fast atom bombardment massspectroscopy.

M+H⁺ Calculated=1093 Found=1093

Preparation A

N-Phenylcarbamylstaurosporine (PCS)

Reference: U.S. Pat. No. 5,093,330

A solution of 2.0 mg of staurosporine (4.30 micromoles) in 1.50 ml ofdried ethyl acetate was treated with 468 μl (4.30 micromoles) of asolution of 10 μl of phenyl isocyanate in 0.990 ml of dried ethylacetate. The solution was allowed to stand overnight and 3 ml of hexanewas added in portions. Colorless crystals were obtained which weighed2.39 mg. After recrystallizing this product from 1 ml of ethyl acetateand 2 ml of petroleum ether, 1.75 mg of a crystalline product wasisolated. From a similar preparation, the product's composition asN-phenylcarbamylstaurosporine was verified by FAB-MS.

M+H⁺ Calculated=586 Found=586

Preparation B

N-Phenylthiocarbamylstaurosporine (PTCS)

A solution of 1.0 mg (2.15 micromoles) of staurosporine in 1.00 ml ofethyl acetate was treated with 26 microliters of a stock solution of 10microliters of phenyl isothiocyanate in 1.00 ml of ethyl acetate. Thisaliquot contained 290 micrograms (2.15 micromoles) of phenylisothiocyanate. The reaction mixture was held at 25° C. overnight, andthen 2.0 ml of hexane was added. The resulting crystalline product wasfiltered off, washed with hexane and dried with a stream of argon gas.

FAB-MS Calc: M+H⁺ =602 Found=602

Preparation C

N-Ethylcarbamylstaurosporine (ECS)

A solution of 0.9 mg (1.93 micromoles) of staurosporine in 900microliters of ethyl acetate was treated with 1.93 micromoles (30.2microliters of a stock solution of 9.05 mg of ethyl isocyanate in 2.00ml of dried ethyl acetate) of ethyl isocyanate. The reaction mixture washeld at 25° C. overnight, and 2.0 ml of hexane was added. Thecrystalline product was separated and dried. ##STR63##

EXAMPLE 3

Compound II-4

Compound A (962 mg, 2 mmol) was dissolved in a mixture of 30 ml oftetrahydrofuran and 10 ml of methanol, and then 760 mg of sodiumborohydride (20 mmol) was added thereto under ice cooling, followed bystirring at the same temperature for 4 hours and further at roomtemperature for 12 hours. After 3N hydrochloric acid was added thereto,the solution was washed with an aqueous solution of sodium chloride anddried over magnesium sulfate, followed by evaporation of the solvent.The residue was purified by silica gel column chromatography(chloroform/methanol=98/2) to give 882 mg (yield 97%) of Compound II-4.

Melting Point: 130°-140° C.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.032(1H, dd, J=5.0, 13.9 Hz), 2.231 (3H, s),2.967(3H, s), 3.609(1H, dd, J=7.6, 13.4 Hz), 3.959(2H, m), 5.000(2H, s),5.268(1H, t, J=5.3 Hz), 7.065(1H, dd, J=4.9, 7.3 Hz), 7.254-8.038 (7H,m), 8.565(1H, s), 9.206(1H, d, J=7.8 Hz) ##STR64##

EXAMPLE 4

Compound II-14

Compound B (393 mg, 0.9 mmol) was dissolved in 25 ml of tetrahydrofuran,and then 3 ml of tetrahydrofuran containing 309 mg ofcarbobenzoxy-L-serine (1.35 mmol), 156 mg of N-hydroxysuccinimide (1.35mmol), 0.1 ml of 4-methylmorpholine (0.9 mmol) and 279 mg ofdicyclohexylcarbodiimide (1.35 mmol) was added under ice cooling,followed by stirring for 12 hours. The reaction mixture was filtered andthe solvent was evaporated. The residue was purified by silica gelcolumn chromatography (chloroform/methanol=99/1) to give 429 mg (yield72%) of Compound C.

Melting Point: 188°-193° C.

SIMS (m/z): 660 (M+1)⁺

Compound C (399 mg) was dissolved in 10 ml of dimethylformamide, andthen 300 mg of 10% palladium on carbon was added, followed by stirringat 50° C. for 7 hours in a hydrogen stream. The reaction mixture wasfiltered through Celite and the solvent was evaporated. The residue waspurified by silica gel column chromatography (chloroform/methanol/28%ammonium hydroxide=90/10/1) and the obtained product was dissolved in 5ml of tetrahydrofuran, followed by addition of 5 ml of 1.7N hydrogenchloride/ethyl acetate and 10 ml of diethyl ether. The precipitate wasseparated from the solution by filtration to give 234 mg (yield 69%) ofCompound II-14.

Melting Point: >300° C.

¹ H-NMR (DMSO-d₆ +D₂ O) δ (ppm): 1.92-2.28(1H, m), 2.20 (3H, s),2.84-3.12(7H, m), 3.40-4.20(5H, m), 5.04 (2H, s), 6.98(1H, m),7.24-8.20(7H, m), 8.76(1H, brs), 9.22(1H, d, J=8 Hz)

SIMS (m/z): 527 (M+2)⁺ ##STR65##

EXAMPLE 5

PC-12 cells are a clonal population arising from a tumor of rat adrenalmedulla, and have proven to be an extremely useful and widely studiedmodel for study of the actions of NGF (Guroff, Cell Culture in theNeurosciences, Plenum Publishing Corporation, pages 245-272, 1985). Oneparticularly robust effect of NGF on these cells is a rapid stimulationof the activity of ornithine decarboxylase (ODC), an effect which wasreported to be blocked by 200 nM K-252a (Koizumi et al., 1988). In theexperiments of this Example, PC-12 cells (obtained from Dr. G. Guroff,National Institute of Health, Bethesda, Md.) were cultured in 48-wellplates at a density of 6×10⁴ cells/cm² and incubated with drug vehicle(0.5% DMSO), K-252a, staurosporine, or HBCS. K-252a and staurosporineare commercially available from Kamiya Biomedical. Four hours after drugaddition, the cells were harvested for ODC assay, as described by Huffet al. (J. Cell Biol. 88: 189-198, 1981).

All three compounds produced an induction (i.e., an increase) of ODCactivity, but there were considerable differences in potency andefficacy (FIG. 1). K-252a produced a dose-dependent induction of ODCactivity, with effects detectable at 2 nM and increasing to a maximum at200 nM (36.3 fold induction). The effects of staurosporine were likewisedetectable at 2 nM, but peaked at 20 nM (34.7 fold induction), anddeclined considerably at 200 nM. HBCS (Example 1) similarly induced at 2nM, but higher concentrations failed to yield an increased effect, sothat the maximum efficacy was much less than that of the other twocompounds (6.5 fold induction). In another experiment, the effects ofPTCS, PCS, and ECS (Example 2) on PC-12 cell ODC activity were comparedto that of K-252a. At 200 nM concentrations, expressing the activity ofK-252a as 100%, PTCS exhibited 71.4% of the activity of K-252a, whilePCS and ECS exhibited 88.9% and 61.9% of the activity of K-252a,respectively. However, the protein kinase C inhibitor H-7 did not induceODC activity at 30 μM, a concentration known to inhibit protein kinase Cactivity (Nakadate et al., Biochem. Pharmacol. 37: 1541-1545, 1988).

The ability of K-252a, staurosporine and HBCS to potentiate and/orinhibit NGF bioactivity was assessed by adding 10 ng NGF per ml of cellculture medium, in the absence or presence of the above compounds in theconcentrations previously indicated, followed by ODC assay of the cellsas described above (FIG. 2). This concentration of NGF was selected toprovide an intermediate level of induction so that either potentiatingor inhibiting effects of the compounds could be detected. K-252a at 200nM inhibited the NGF induction of ODC, as reported by Koizumi et al.(1988), but, surprisingly, potentiated the induction at lowerconcentrations (2 nM and 20 nM). Staurosporine, at 2 nM, alsopotentiated the induction by NGF, but this effect was lost at higherconcentrations (20 and 200 nM). HBCS, in contrast, potentiated theeffects of NGF at all concentrations tested. This striking effect isshown relative to the modest ODC-inducing effects of HBCS alone in FIG.3.

EXAMPLE 6

The effect of K-252a on choline acetyltransferase (CHAT) activity wasassayed in dissociated spinal cord cultures prepared from fetal rats bystandard methods (see below). ChAT is the enzyme that catalyzes thesynthesis of the neurotransmitter acetylcholine, and is a specificbiochemical marker for cholinergic neurons. In the spinal cord, thelarge majority of cholinergic neurons are motor neurons. Assay of thisenzyme may thus be used as an indication of the effects of a factor (orfactors) on the survival of cholinergic neurons and/or regulation ofthis enzyme.

K-252a was added at the indicated concentrations to the cultures afterincubating 2-3 hours after plating to allow cells to attach to thesubstrate. ChAT activity was measured after 48 hours in culture. K-252ain spinal cord cultures resulted in a dose dependent increase in CHATactivity with maximum efficacy (2- to 3-fold increase) achieved at200-300 nM (FIG. 4). Higher concentrations resulted in a decrease inChAT activity (FIG. 4). Longer culture incubation times, up to sevendays, resulted in 4-to-5 fold increases in ChAT activity (FIG. 5) due tothe decreased basal level of ChAT activity. In this culture system,increasing numbers of motor neurons degenerate and die over time underbasal (control) conditions (McManaman et al., Developmental Biol.125:311-320, 1988). The results shown in both FIGS. 4 and 5 are theresult of a single application of K-252a on the day of cultureinitiation, indicating a prolonged effect on the survival of spinal cordcholinergic neurons and/or regulation of the enzyme itself.

Experiments with dissociated cultures of fetal rat spinal cord cellswere performed generally as described (Smith et al., J. Cell Biol.101:1608-1621, 1985). Dissociated cells were prepared from spinal cordsdissected from day 14 embryonic rats by standard techniques known tothose skilled in the art, using trypsin dissociation of tissue (Smith etal., supra). Cells were seeded (plated) at 6×10⁵ cells/cm² inpoly-1-ornithine coated plastic tissue culture wells in serum-free N2medium and incubated at 37° C. in a humidified atmosphere of 5% CO₂ /95%air (Bottenstein et al., Proc. Natl. Acad. Sci. USA 76:514-517, 1979)for 48 hours. ChAT activity was measured using modifications of theFonnum procedure (J. Neurochem. 24:407-409, 1975) according to Ishidaand Deguchi (J. Neurosci. 3:1818-1823, 1983), and McManaman et al.,supra (1988). Activity was normalized to total protein measured by thebicinchonicic acid/Cu⁺⁺ reaction (BCA protein assay reagent, Pierce,Rockland, Ill.).

EXAMPLE 7

Over one hundred functional derivatives of K-252a were tested in thespinal cord CHAT assay to determine their relative efficacy. The data inFIG. 8 show that of the original functional derivatives tested at 300and 30 nM, 28 resulted in significantly increased ChAT activity at 300nM. One functional derivative, compound II-21, was also active at 30 nM(30% enhancement of ChAT activity over basal levels). This compound wasmore potent than K-252a or the remaining analogs since none of theseactively enhanced ChAT activity at 30 nM.

FIG. 13a shows the ability of the original 28 K-252a derivatives shownto significantly increase ChAT activity in rat spinal cord cultures, aswell as 30 additional derivatives (compounds II-29 through II-34, II-36through II-56, and IV-1 through IV-3, all inclusive). FIG. 13b shows theability of K-252a derivatives II-66-80, IV-5, IV-6, VI-1, and VI-2 tosignificantly increase ChAT activity in rat spinal cord cultures. FIG.13C shows the ability of 12 additional K252a derivatives tosignificantly increase ChAT activity in rat spinal cord cultures.

EXAMPLE 8

K-252a as well as over 50 functional derivatives were assessed for theirability to promote dorsal root ganglion neuron cell survival. Cellsurvival was measured by uptake of calcein AM, an analog of the viabledye, fluorescein diacetate. Calcein is taken up by viable cells andcleaved intracellularly to fluorescent salts which are retained byintact membranes of viable cells. Microscopic counts of viable neuronscorrelate directly with relative fluorescence values obtained with thefluorimetric viability assay. This method thus provides a reliable andquantitive measurement of cell survival in the total cell population ofa given culture (Bozyczko-Coyne et al., J. Neur. Meth. 50:205-216,1993).

Dorsal root ganglia were dissected from embryonic age day 8 chickembryos and dissociated cells prepared by subsequent Dispase (neutralprotease, Collaborative Research) dissociation. Neurons were seeded atlow density (1.8×10⁴ cells/cm²) into 96 well poly-L-ornithine andlaminin coated plates. Cells were cultured for 48 hours in serum-free N2medium (Bottenstein and Sato, 1979) at 37° C. in a humidifiedatmosphere, 5% Co₂ /95% air. Cell survival was assessed at 48 hoursusing the viable fluorimetric assay described above.

Dorsal root ganglion neuron survival was enhanced by K-252a in aconcentration-dependent manner. Maximum activity was observed atapproximately 100 nM (FIG. 6). Twenty-four of the 50 analogs tested wereactive in promoting DRG neuron survival, twenty-two of which are shownin FIG. 7. All of these analogs were also active in increasing spinalcord CHAT activity (see Example 5, FIG. 8). The original 22 as well asthe 2 additional active analogs (II-30, II-32) are shown in FIG. 14.Microscopic examination of the dorsal root ganglion neurons stimulatedwith the twenty-four active functional derivatives indicated enhancednerve fiber outgrowth as well.

EXAMPLE 9

Infusion of the excitatory amino acid kainic acid (kainate) directlyinto the ventricles of a rodent brain results in neuronal degenerationof the pyramidal cells of the hippocampus. This neuronal death ischaracterized by a marked increase in the proteolysis of thecytoskeletal protein, spectrin. Spectrin breakdown products can bemeasured in homogenates of the hippocampus within 24 hours followingkainate administration. The magnitude of spectrin proteolysis is highlycorrelated with the magnitude of neuronal death in pyramidal cells ofthe hippocampus (Siman et al., J. Neurosci. 9:1579-1590, 1989), and thusspectrin proteolysis is an excellent biochemical marker of excitatoryamino acid-induced neuronal degeneration. Excessive release ofendogenous excitatory amino acids has been implicated as an etiology innumerous neurological diseases and disorders, including stroke and otherischemic injuries; Alzheimer's disease; motor neuron disease includingamyotrophic lateral sclerosis; Parkinson's disease; Huntington'sdisease; AIDS dementia; epilepsy; and concussive or penetrating injuriesof the brain or spinal cord.

The results shown in FIGS. 9-12 were generated according to thefollowing methods:

Kainate infusion regime:

The effect of K-252a or its derivatives on kainate-induced neuronaldamage was evaluated. Adult male or female Sprague-Dawley rats (175-250g) were anesthetized with Nembutal (50 mg/kg, ip). Each rat wasadministered a test compound (in a total of 5 μl) before and afterkainate treatment (5 μl) by icv infusion. This was done using a dose andinfusion schedule as indicated for individual cases above. Controlanimals received vehicle instead of kainate and drug infusion. Foranatomical studies, icv infusions were delivered through a cannula(Plastic One, Roanoke, Va.) implanted approximately one week before druginfusions, and positioned at stereotaxic coordinates: anterior-posteriorat bregma, 1.5 mm lateral to bregma, and 4.4 mm ventral from the top ofthe skull. Results of this treatment regimen were evaluated two weekslater using the anatomical analysis described below.

In studies to assess the effect of K-252a or its derivatives onkainate-induced spectrin proteolysis, anesthetized rats received a 5 μlicv infusion of the drug, or vehicle, simultaneously with kainate,through a 10 μl Hamilton syringe positioned at the stereotaxiccoordinates described above. These rats were killed 24 hours later andsubjected to biochemical analysis as described below.

Anatomical and Biochemical Analyses:

Anatomical analysis was performed as follows. Rats were killed bydecapitation 2 weeks following treatments, and the brains were rapidlyremoved and frozen on dry ice. A series of slide-mounted coronalsections from each brain was stained with thionin and examinedmicroscopically. Damage to the hippocampus was quantified by summing thetotal number of 4 anatomically defined regions of the hippocampus (CA1-4according to the classification of Lorente de No, as described byShepard, 1979, The Synaptic Organization of the Brain, Oxford, p. 310,hereby incorporated by reference), on both left and right sides of thebrain, that suffered a loss of pyramidal cells.

Biochemical analysis was performed as follows: Calpain I-sensitiveproteolysis of brain spectrin (fodrin) was evaluated in homogenates ofthe hippocampus using an immunoblot analysis described by Siman et al.(1988, Neuron, 1: 279-287, hereby incorporated by reference). Briefly,rats were killed by decapitation 24 hours following treatment, and thedorsal hippocampus was rapidly dissected out of the brain andhomogenized in 20 mM Tris-HCl (pH 7.4) containing 0.1 mMphenylmethylsulfonyl fluoride. Proteins from aliquots of each homogenatewere separated by SDS-PAGE, and an immunoblot analysis was used toquantitate the amount of kainate-induced spectrin breakdown in eachsample.

FIG. 9 shows the effect of K-252a on kainate-induced neuronaldegeneration in the hippocampus. Cannulated male and femaleSprague-Dawley rats received 0.4 μg of K-252a, or vehicle, 30 minutesprior to and about 3 and 24 hours following kainate (0.6 μg) injectiondirectly into the lateral cerebral ventricles of the brain (icv). Twoweeks later the brains were excised, frozen, sectioned, and stained forhistological analysis, as described below. Data shown are the meannumber of sub-regions of the hippocampus damaged for each group,±Standard Error of the Means (S.E.M.). K-252a significantly reduced thenumber of damaged areas within the hippocampus from 3.86±0.78 (in theabsence of K-252a) to 1.18±0.4 (in the presence of K-252a).

FIG. 10 shows the effect of K-252a on kainate-induced spectrin breakdownin the hippocampus. Female Sprague-Dawley rats received 0.4 μg ofK-252a, or vehicle, together with a neurotoxic dose of kainate (0.6 μg),by icv infusion. Sham control animals received infusions of vehicle, butno kainate or K-252a. Twenty-four hours later, homogenates of the dorsalhippocampus were analyzed for spectrin breakdown products as describedbelow. The magnitude of spectrin proteolysis is expressed as a percentincrease in spectrin breakdown products for each group over sham controlvalues. Data shown are the mean percent increase in spectrin breakdownproducts for each group (sham =100%)±S.E.M. Icv infusion of K-252asignificantly reduced the extent of spectrin proteolysis, from about140±15% (in the absence of K-252a) to approximately 102±10% (in thepresence of K-252a) of sham values.

FIG. 11 shows the effect of HBCS on kainate-induced neuronaldegeneration in the hippocampus. Cannulated female Sprague-Dawley ratsreceived 0.8 μg of HBCS, or vehicle, 40 minutes prior to and about 4hours following kainate (0.6 μg) by icv infusion. Two weeks later thebrains were excised, frozen, sectioned and stained for histologicalanalysis, as described below. Data shown are the mean number ofsub-regions of the hippocampus damaged for each group, ±S.E.M. HBCSsignificantly reduced the number of damaged areas within the hippocampusfrom about 2.5±0.6 (without HBCS treatment) to 1.3±0.5 (with HBCStreatment).

FIG. 12 compares the effect of three K-252a functional derivatives onkainate-induced spectrin breakdown in the hippocampus. FemaleSprague-Dawley rats received 0.4 μg of K-252a, or compounds III-1, orII-21, or vehicle, together with a neurotoxic dose of kainate (0.6 μg),by icv infusion. Sham control animals received infusions of vehicle, butno kainate or K-252a derivative. Twenty-four hours later, homogenates ofthe dorsal hippocampus were analyzed for spectrin breakdown products asdescribed below. The magnitude of spectrin proteolysis is expressed as apercent increase in spectrin breakdown products for each group over shamcontrol values. Data shown are the mean percent increase in spectrinbreakdown products for each group (sham=100%)±S.E.M. Icv infusion ofK-252a reduced the extent of spectrin proteolysis, from about 128±9%(vehicle treatment) to approximately 104±4% (in the presence of K-252a)of sham values. K-252a derivatives III-1 and II-21 failed to preventkainate-induced spectrin proteolysis.

EXAMPLE 10

K-252a was assayed for the ability to promote survival in striatalcultures. Striata were dissected from embryonic day 17 rat embryos andcells were dissociated by Dispase (neutral protease, CollaborativeResearch). Neurons were seeded at 5×10⁴ cells/well (1.5×10⁵ /cm²) in96-well plates, the wells having been previously coated withpoly-1-ornithine and laminin. Cells were cultured in serum-free N2medium containing 0.05% bovine serum albumin (Bottenstein and Sato,1979) at 37° C. in a humidified atmosphere, 5% CO₂ /95% air. Cellsurvival was assayed 5 days after seeding using the calcein viable cellfluorimetric assay described in Example 8.

The survival of striatal neurons was enhanced by K-252a in aconcentration-dependent manner. Maximum activity was found with 75 nMK-252a, which produced an efficacy of 3-4 fold over control (FIG. 15).In the control cultures, 90% of the neurons plated on day 0 died within5 days, whereas in cultures treated with K-252a, 50% of the neuronssurvived (FIG. 16). The survival effect in striatal neurons occurredafter 3 days in culture and was sustained for at least 7 days inculture. These results are from a single application of K-252a on theday of culture initiation, and indicate that the effect on neuronsurvival is sustained.

FIG. 17 is a pair of photomicrographs taken from control cultures orcultures treated with 75 nM K-252a. There was an increase in cellsurvival and neurite outgrowth in these cultures in the presence of 75nM K-252a.

EXAMPLE 11

Thirty-one functional derivatives of K-252a were tested to determinetheir potency and efficacy in the striatal cell survival assay ofExample 10. FIG. 18 shows data on 18 K-252a derivatives that promotedthe survival of striatal neurons.

EXAMPLE 12

Compounds of the invention were assessed for their ability to promotesurvival and increase ChAT activity in basal forebrain cultures. ChATactivity in these cultures is a biochemical marker for the cholinergicneurons (less than 5% of the cells in culture), which represent themajor cholinergic input to the hippocampal formation, olfactory nucleus,interpeduncular nucleus, cortex, amygdala, and parts of the thalamus.Representative compounds of the invention not only increased ChATactivity but in addition increased general survival of neurons in basalforebrain cultures.

The basal forebrain was dissected from embryonic day 17 or 18 ratembryos and the cells were dissociated with Dispase™ (neutral protease,Collaborative Research). Neurons were plated at 5×10⁴ cells/well(1.5×10⁵ cells/cm²) in wells of 96-well plates previously coated withpoly-1-ornithine and laminin. Cells were cultured in serum-free N₂medium containing 0.05% bovine serum albumin (BSA) (Bottenstein et al.,supra) at 37° C. in a humidified atmosphere of 5% CO₂ /95% air. ChATactivity was measured in vitro at day six, using a modification of theFonnum procedure (supra) according to McManaman et al. (supra) andGlicksman et al. (J. Neurochem. 61:210-221, 1993). Cell survival wasassessed 5 days after plating using the calcein AM fluorimetric assaydescribed by Bozyczko-Coyne et al. (supra). Culture medium was partiallyaspirated at the time of assay to leave 50 microliters per well. EightμM calcein AM stock in 150 μl of Dulbecco's phosphate buffered saline(DPBS; Gibco BRL) was then added to give a final concentration of 6 μM,in 200 μl per well, in a 96-well plate. The plates were incubated for 30minutes at 37° C., followed by four serial dilutions with 200 μl DPBS.The relative fluorescence of each well was measured using aplate-reading fluorimeter (Cytofluor 2350, Millipore) at an excitationwavelength of 485 nm, an emission wavelength of 538 nm, and asensitivity setting of #3. The mean fluorescence background calculatedfrom six wells that received calcein AM but contained no cells wassubtracted from all values. The linearity of the fluorescence signal wasverified for the 30 minute substrate incubation time for the range ofcell densities encountered in these experiments. K-252a, as well as atleast twelve K-252a derivatives (II-3, II-5, II-10, II-20, II-21, II-22,II-30, II-32, II-51, II-62, II-63, II-64, II-65) promoted the survivalof basal forebrain neurons (FIG. 19).

EXAMPLE 13

The following tests were conducted to evaluate the effect of CompoundII-51 on cortical cholinergic function when rats were subjected tolesions of the nucleus basalis.

Cholinergic neurons originating from the basal forebrain and projectingto the hippocampus via the septo-hippocampal pathway, or to the cortexvia the basalo-cortico pathway, undergo profound degeneration during thecourse and progression of Alzheimer's disease. There is some degree ofcorrelation between loss of these neurons and decreases in cognitive andmemory function in individuals afflicted with this disorder (Fibiger, H.Trends in Neurosci 14:220-223, 1991). Several models of cholinergicdysfunction have been proposed which show loss of biochemical markers aswell as behavioral deficits. These models parallel the progression ofAlzheimer's disease (Olton, D. et al, "Dementia: animal models of thecognitive impairments produced by degeneration of the basal forebraincholinergic system" in Meltzer, H., (Ed.) Psychopharmacology: The ThirdGeneration of Progress, Raven Press, N.Y., 1987, pp. 941-953; Smith, S.,Brain Res. Rev. 13:103-118, 1988). For example, one model of cholinergicdegeneration is excitotoxic lesioning of the nucleus basalis (Wenk. G.et al., Exp. Brain Res. 56:335-340, 1984). Lesions in cholinergicneurons within the basal forebrain result in loss of neuronal cellbodies in this region, and subsequent loss of cholinergic terminalmarkers in the frontal and parietal cortex (Dunnett, S. et al. TrendsNeurosci 14:494-501, 1991). Using the following methods, Compound II-51was shown to increase cortical cholinergic function in rats that weresubjected to lesions of the nucleus basalis.

Male Sprague-Dawley rats (225-275 grams) were used for all experiments.Unilateral ibotenic lesions of the nucleus basalis magnocellularis wereproduced by methods known to those of skill in the art (see, e.g., Wenket al. supra), with modifications as described below. Rats wereanesthetized with 50 mg/kg pentobarbital and 5 μg of ibotenic acid (in 1μl of PBS) was injected, unilaterally, into the nucleus basalismagnocellularis. The coordinates used were from the Paxinos and Watsonbrain atlas (1.5 mm posterior, 2.8 mm lateral, 7.2 mm dorso-ventral).Injections took place over a period of 6 minutes. Dye injectionsindicated that the injections went directly into the nucleus basalis.

Compound II-51 was dissolved in 30% Solutol™ at concentrations of 0.01to 0.3 mg/ml. The compound (or the Solutol™ vehicle) was administeredsubcutaneously one day after, or 6 hours prior to, inducing lesions inthe nucleus basalis, and every 48 hours thereafter. Doses were 0.01,0.03, 0.10 and 0.30 mg/kg. Experiments were terminated from 4 to 21 daysafter inducing lesions. ChAT activity was measured in tissue homogenatesof the frontal-parietal cortex by the method of Fonnum (supra). ChATactivity in the frontal cortex, ipsilateral to the side of the lesionwas compared and normalized to ChAT activity on the contralateral(lesion-free side). CHAT activity is expressed as the ipsilateral tocontralateral ratio.

The data were analyzed by ANOVA and differences between treatmentsdetermined by post-hoc Tukey's test. Means were considered significantlydifferent if p<0.05.

In animals in which lesions were made in the nucleus basalis, there wasa time dependent decrease in cortical ChAT activity with maximum lossdacurring between 3 and 7 days after lesion (Table 6). Route ofadministration, doses and dosing schedule were based on preliminary datashowing the effects of Compound II-51 on ChAT levels in the basalforebrain of adult rats. To assess the effects of Compound II-51 on ChATlevels (i.e., on cholinergic function) in animals with lesions, the drugwas administered one day after inducing lesions for 14 to 21 days, or 6hours prior to surgery for 4 days.

                  TABLE 6                                                         ______________________________________                                        Time Course of Loss of Cortical ChAT Activity after                           Inducing Unilateral Lesions in the Nucleus Basalis Magnocellularis.sup.a                                  ChAT Activity                                     Lesion Time (hrs.)                                                                          Injection     (Ipsi/Contra ratio)                               ______________________________________                                        No lesion Control                                                                           --             96 ± 8                                         8 hrs.       Ibotenic acid (5 μg)                                                                      97 ± 14                                        24 hrs.      Ibotenic acid (5 μg)                                                                     105 ± 11                                        72 hrs.      Ibotenic acid (5 μg)                                                                      74 ± 11.sup.b                                 168 hrs. (7 days)                                                                           Ibotenic acid (5 μg)                                                                      70 ± 7.sup.b                                  ______________________________________                                         .sup.a Unilateral lesions were induced in the NBM of rats. Frontal cortex     was assayed for ChAT activity at indicated time after lesion.                 .sup.b Indicates significantly different from lesionfree control (p <         0.05).                                                                   

Dose-response studies with Compound II-51 were conducted at doses of0.01, 0.03 and 0.10 mg/kg (Table 7). Subcutaneous injections of CompoundII-51 were given on alternate days for 21 days, starting one day afterinducing lesions with Ibotenic acid. Results showed that at a dose aslow as 0.03 mg/kg, Compound II-51 was effective in attenuating thedecease in cortical CHAT activity (Table 7).

                  TABLE 7                                                         ______________________________________                                        Effects of Systematically Administered Compound II-51 on Cortical             ChAT Activity in NBM Rats with Lesions: Dose-Response Study.sup.a                                         ChAT Activity                                     Lesion Treatment                                                                           Dose Compound II-51                                                                          (Ipsi/Contra ratio)                               ______________________________________                                        No lesion    --             98.4 ± 4.5                                     With lesion  Vehicle        67.4 ± 7.2.sup.b                               With lesion  0.01 mg/kg QOD 70.1 ± 11.2                                    With lesion  0.03 mg/kg QOD 93.8 ± 14.9.sup.c                              With lesion  0.10 mg/kg QOD 87.9 ± 11.6.sup.c                              ______________________________________                                         .sup.a Unilateral lesions were induced in the NBM of rats. Twentyfour         hours after inducing lesions, subcutaneous administration of Compound II5     commenced. Twentyone days after lesioning animals were sacrificed and         cortical ChAT activity assessed.                                              .sup.b Indicates significantly different than control (p < 0.05).             .sup.c Indicates significantly different than lesion alone.              

Systemic administration of Compound II-51 attenuated the decrease incholinergic function in the frontal cortex measured at 4, 14 and 21 daysafter inducing lesions (Table 8). In rats with unilateral lesions, ChATactivity on the contralateral side was unchanged, suggesting thatCompound II-51 only affected neurons with lesions.

                  TABLE 8                                                         ______________________________________                                        Effects of Systematically Administered Compound II-51 on Cortical             ChAT Activity in NBM Rats with Lesions: Time-Response Study.sup.a                                        ChAT Activity                                      Lesion Time (Days)                                                                        Dose Compound II-51                                                                          (Ipsi/Contra ratio)                                ______________________________________                                        No lesion Control                                                                         --             99 ± 6                                           4 days     Vehicle        77 ± 6.sup.b                                                0.1 mg/kg (QOD)                                                                              96 ± 12.sup.c                                   14 days     Vehicle        72 ± 8.sup.b                                                0.1 mg/kg (QOD)                                                                              94 ± 6.sup.c                                    21 days     Vehicle        66 ± 8.sup.b                                                0.1 mg/kg (QOD)                                                                              87 ± 7.sup.c                                    ______________________________________                                         .sup.a Unilateral lesions were induced in the NBM of rats. Six hours prio     or 1 after inducing lesions, subcutaneous administration of Compound II51     commenced. Frontal cortex was assayed for ChAT activity at indicated time     after lesion.                                                                 .sup.b Indicates significantly different from control (p < 0.05).             .sup.c Indicates significantly different from lesion + vehicle at same        time point.                                                              

EXAMPLE 14

An in ovo model can be used to evaluate the ability of a compound toinfluence developmentally programmed motoneuron death.

In the chick, somatic motoneurons undergo naturally occurring deathbetween embryonic days 6 and 10 (E6 and E10) (Chu-Wang et al., J. Comp.Neurol. 177:33-58, 1978; Hamburger, J. Comp. Neurol. 160:535-546, 1975;McManaman et al., Neuron 4:891-898, 1990). During this period, thenumber of motoneurons on the two sides of the lumbar spinal cord ofdeveloping chick embryos is reduced from about 46,000 to 23,000.

Chick embryos (E6-E9) were treated with either vehicle (5% Solutol™ HS15, BASF Aktiengesellschaft) or concentrations of Compound II-51 asdescribed. The treatments (50 μl) were applied to the vascularizedchorioallantoic membrane through a window in the shell by the method ofOppenheim et al. (Science 240:919-921, 1988). Embryos were sacrificed onE10 and spinal chords were removed, fixed in Carnoy's solution (10%acetic acid, 60% ethanol, 30% chloroform), embedded in paraffin,sectioned into 8 μm sections, and stained with thionin as describedpreviously (Oppenheim et al., supra). Motoneurons (identified bymorphology and position) were counted blind in every tenth sectionaccording to previously established criteria (Oppenheim et al., J. Comp.Neurol. 210:174-189, 1982; Oppenheim et al., J. Comp. Neurol.246:281-286, 1986).

Daily application of compound II-51 to the chorioallantoic membrane ofE6 to E9 chicks in ovo resulted in a dose-dependent increase in thenumber of surviving lumbar motoneurons (FIG. 20). At the lowesteffective dose tested (1.17 μg/egg) there was a 16% enhancement inmotoneuron survival. The maximal effect was achieved at a dose of 2.3μg/egg, resulting in a 40% increase in motoneuron survival in treatedversus control, vehicle-treated, embryos. At 7 μg/egg there was nofurther increase in motoneuron survival, indicating that in thissituation a maximal response had been achieved at 2.3 μg/egg.

EXAMPLE 15

Motoneurons in the hypoglossal nucleus innervate the tongue via thehypoglossal nerve. In adult rats, transection of the hypoglossal nerveresults in a dramatic loss of ChAT activity in the motoneurons of thehypoglossal nucleus without affecting cell number. The loss of ChATactivity serves as a model for reversion to an immature phenotype.

The left hypoglossal nerve was cut under the Dawleydigastric muscle ofthe neck of each adult female Sprague- rat (120-180g) under Nembutalanesthesia. Fifty microliters of compound II-51 in 10% Solutol™ (HS 15,BASF Aktiengesellschaft) or vehicle alone was applied to a piece ofgelfoam, then the proximal end of the transected nerve together with thegelfoam was wrapped in parafilm. At the end of 7 days, rats wereperfused under deep anesthesia with 4% paraformaldehyde in Sorenson'sbuffer (0.1M NaPO₄, pH 7). The brainstem was removed and immersed infixative for 24 hours, then rinsed well and placed in 30% sucrose forcryoprotection prior to sectioning. Forty micron coronal sections of thebrain were cut and stored in Sorenson's buffer at 4° C. until stained.The hypoglossal nucleus spanned 40-48 sections and every fifth sectionwas processed for immunohistochemistry using the anti-ChAT mousemonoclonal antibody, 1E6, as previously described (Chiu et al., J. Comp.Neurol. 328:351-363, 1993). ChAT immunoreactive neurons were visualizedin sections by avidin-biotin amplification with the Vectastain EliteABC™ kit (Vector Laboratories, Burlingame, Calif.).

Every fifth section from the hypoglossal nucleus was processed andimmunoreactive cells in the control (uninjured) and axotomized sides ofeach animal were counted. Results are expressed as the percentage ofChAT-immunoreactive neurons on the axotomized side in relation to thenumber of ChAT-immunoreactive neurons on the uninjured side. Applicationof 100 μg of compound II-51 to the cut end of the hypoglossal nerveresulted in a significant number of ChAT-immunoreactive neurons(33.7%±9.9 (mean±SEM)) (FIG. 21). In contrast, there were very fewChAT-immunoreactive neurons (8.07%±2.9 (mean±SEM)) in thevehicle-treated control animals.

Preparatory Methods EXAMPLE 16

Compounds (V)

The processes for producing Compounds (V) are described below.

Process 1

Compound (V-1) (examples of Compound (V) in which R¹ is CH₂ So₂ R⁷ and Xis CO₂ R⁵) can be prepared by the following reaction step: ##STR66##

(R⁵ represents lower alkyl or CH₂ NHCO₂ R⁶ in which R⁶ represents loweralkyl or aryl; R⁷ represents lower alkyl.)

The starting compound (A) is disclosed in Japanese Published UnexaminedPatent Application No. 295588/88 (hereby incorporated by reference).

Compound (V-1) can be obtained by treatment of Compound (A) with 1 to1.5 equivalents of an oxidant. An example of the oxidant ism-chloroperbenzoic acid. As a reaction solvent, a halogenatedhydrocarbon such as methylene chloride, chloroform, or ethylenedichloride, or the like is used. The reaction is completed in 0.5 to 1hour at -20° to 30° C.

Process 2

Compounds of Formula (V-2) (examples of Compound (V) in which R¹ ishydrogen and X is CH₂ NHCO₂ R⁶ ! can be prepared by the followingreaction step: ##STR67##

R⁶ represents lower alkyl or aryl.

The starting compound (B) is disclosed in Japanese Published UnexaminedPatent Application No. 155285/87 (hereby incorporated by reference).

Compound (V-2) can be obtained by reaction of Compound (B) with 1 to 3equivalents ClCO₂ R⁶ in the presence of 1 to 3 equivlents of a base. Anexample of the base is triethylamine. As a reaction solvent, ahalogenated hydrocarbon such as methylene chloride, chloroform, orethylene dichloride, or the like is used. The reaction is completed in0.5 to 3 hours at -10° to 30° C.

EXAMPLE 17

Compound II-49

Compound (A; R⁵ ═CH₃ and R⁷ ═C₂ H₅) (27 mg, 0.05 mmol) was dissolved in1 ml of chloroform, and then 10 mg (0.06 mmol) of m-chloroperbenzoicacid was added thereto under ice cooling, followed by stirring at thesame temperature for 45 minutes. After dilution with chloroform, themixture was washed successively with a 8% aqueous solution of sodiumthiosulfate, a saturated aqueous solution of sodium bicarbonate, water,and a saline solution, and dried over sodium sulfate. After evaporationof the solvent, the residue was subjected to silica gel columnchromatography (chloroform/methanol=95/5) to give 17.7 mg (yield 62%) ofCompound II-49.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.298(3H, t, J=7.5 Hz), 2.037 (1H, dd, J-5.0,14.1 Hz), 2.153(3H, s), 3.096(2H,q, J=7.5 Hz), 3.266 (2H, s), 3.929(3H,s), 4.985 (1H, d, J=17.0 Hz), 5.043(1H, d, J=17.0Hz), 6.348(1H, s),7.147 (1H, dd, J=4.9, 7.1 Hz), 7.345-8.070(6H, m), 8.612(1H, s),9.232(1H, d, J=1.5 Hz)

FAB-MS (m/z): 574 (M+1)⁺

EXAMPLE 18

Compound II-57

Compound (B) (43.8 mg, 0.1 mmol) was dissolved in 1 ml oftetrahydrofuran, and then 9.3 μl (0.12 mmol) methyl chloroformate and 28μl(0.12 mmol) of triethylamine were added thereto, followed by stirringfor 50 minutes under ice cooling. After dilution with tetrahydrofuran,the mixture was washed with a saline solution, and dried over sodiumsulfate. After evaporation of the solvent, the residue was subjected tosilica gel column chromatography (chloroform/methanol=99/1) to give 32.6mg of Compound II-57. ¹ H-NMR (CDCl₃) δ (ppm): 2.099(3H, s), 2.679(1H,m), 3.204(1H, dd, J=6.7 m 13.8 Hz), 3.837(3H, s), 4.446 (1H, d, J=17.3Hz), 4,634 (1H, d, J=17.6 Hz), 5.497 (1H, brs), 6.591(1H, brs),7.010-8.037(7H, m), 8.592(1H, d, J=6.6 Hz)

FAB-MS (m/z): 497 (M+1)⁺

EXAMPLE 19

Compound II-38

Substantially the same procedure as in Example 18 was repeated using43.8 mg (0.1 mmol) of Compound (B) and 15 μl of phenyl chloroformate togive 27.8 mg (yield 50%) of Compound II-38.

¹ H-NMR (CDCl₃) δ (ppm): 2.111(3H, s), 2.890(1H, brd, J=13.7 Hz),3.262(1H, dd, J=7.5, 13.9 Hz), 3.742(1H, d, J=13.4 Hz), 3.967(1H, d,J=12.9 Hz), 4.582(1H, d, J=16.3 Hz), 5.342(1H, brs), 5.906(1H, brs),6.550 (1H, brs), 7.005-8.042(12H, m), 8.596(1H, d, J=7.6 Hz)

FAB-MS (m/z): 559 (M+1)⁺

EXAMPLE 20

(The synthesis of Compound H from Compound C is shown in FIG. 22.)

Compound II-39

Compound (C) (Japanese Published Unexamined Patent Application No.295588/88; hereby incorporated by reference) (20 mg, 0.035 mmol) wasdissolved in 1 ml of chloroform, and then 14.6 μl (0.105 mmol) oftriethylamine and 13.9 μl (0.175 mmol) of ethyl isocyanate were addedthereto, followed by stirring at room temperature for 2 hours. To thesolution was added 1 ml of methanol, followed by dilution withchloroform. The mixture was washed successively with water and a salinesolution, and dried over sodium sulfate. After evaporation of thesolvent, the residue was subjected to silica gel column chromatography(chloroform/methanol=98/2) to give 21 mg (yield 84% of Compound (D).

¹ H-NMR (CDCl₃) δ (ppm): 1.195(3H, t, J=7.2 Hz), 1.222(3H, t, J=7.2 Hz),1.664(3H, s), 2.194(3H, s), 2.555(3H, s), 3.346(4H, q, J=7.2 Hz),3.820(1H, dd, J=7.5, 14.6 Hz), 3.938(3H, s), 5.036(1H, d, J=17.7 Hz),5.125(1H, d, J=17.2 Hz), 6.745(1H, dd, J=4.8, 7.4 Hz), 7.260-7.898(5H,m), 8.690(1H, d, J=1.9 Hz)

FAB-MS (m/z): 724 (M+1)⁺

Compound (D) (9 mg, 0.012 mmol) was dissolved in a mixture of 0.2 ml oftetrahydrofuran and 0.2 ml of methanol, and then 2 μl of 28% sodiummethoxide/methanol was added thereto, followed by stirring at roomtemperature for 10 minutes. To the solution was added 0.1 ml of a 5%aqueous solution of citric acid, followed by dilution with chloroform.The mixture was washed successively with water and a saline solution,and dried over sodium sulfate. After evaporation of the solvent, theresidue was subjected to silica gel column chromatography(chloroform/methanol=9/1) to give 8 mg of Compound II-39.

1H-NMR (DMSO-d₆) δ (ppm): 1.086(3H, t, J=7.1 Hz), 1.099 (3H, t, J=7.1Hz), 1.948(1H, dd, J=4.8, 14.1 Hz), 2.107(3H, s), 3.158(4H, m),3.910(3H, s), 4.880(1H, d, J=17.7 Hz), 4.931(1H, d, J=16.9 Hz),7.028(1H, dd, J=5.0, 7.1 Hz), 7.332-8.287(5H, m), 8.838(1H, d, J=2.1 Hz)

FAB-MS (m/z): 640 (M+1)⁺

EXAMPLE 21

Compounds II-51 and II-56

Compound (E) (Japanese Published Unexamined Patent Application No.295588/88; supra) (60.7 mg, 0.1 mmol) was dissolved in a mixture of 5 mlof chloroform and 1 ml of methanol, and then 11 mg (0.3 mmol) of sodiumborohydride was added thereto under ice cooling, followed by stirring atthe same temperature for 15 minutes. After dilution with chloroform, themixture was washed successively with water and a saline solution, anddried over potassium carbonate. After evaporation of the solvent, theresidue was subjected to silica gel column chromatography(Chloroform/methanol/triethylamine=98/2/0.5) to give 36 mg (yield 59%)of Compound (F).

¹ H-NMR (DMSO-d₆) δ (ppm): 1.650(3H, s), 2.027(1H, dd, J=4.9, 14.5 Hz),2.126(3H, s), 3.843(1H, dd, J=7.4, 14.5 Hz), 3.891(3H, s), 4.607(2H, s),4.673(2H, s), 5.125(2H, s), 7.099(1H, dd, J=5.0, 7.3 Hz),7.437-7.907(5H, m), 8.812(1H, d, J=0.8 Hz)

FAB-MS (m/z): 612 (M+1)⁺

Compound (F) (159 mg, 0.26 mmol) was dissolved in 15 ml of chloroform,and then 0.8 ml (10.4 mmol) of ethanethiol and 24 mg (0.104 mmol) ofcamphorsulfonic acid were added thereto, followed by stirring at roomtemperature for 12 hours. The solution was washed successively with asaturated aqueous solution of sodium bicarbonate, water, and a salinesolution, and dried over sodium sulfate. After evaporation of thesolvent, the residue was subjected to silica gel column chromatography(ethyl acetate/toluene=1/9-chloroform/methanol=99/1) to give 43 mg ofCompound (G) and 75 mg of Compound (H).

Compound (G)

¹ H-NMR (CDCl₃) δ (ppm): 1.292(3H, t, J=7.4 Hz), 1.297 (3H, t, J=7.4Hz), 1.799(3H, s), 2.141(1H, dd, J=5.0, 14.5 Hz), 2.256(3H, s),2.532(2H, q, J=7.4 Hz), 2.553(2H, q, J=7.4 Hz), 2.869(3H, s), 3.971(1H,dd, J=7.5, 14.5 Hz), 3.992(2H, s), 4.005 (3H, s), 4.021(2H, s),5.416(1H, dd, J=17.5 Hz), 5.459(1H, d, J=17.4 Hz), 6.989(1H, dd, J=5.1,7.4 Hz), 7.509-7.963(5H, m), 9.134(1H, d, J=1.2 Hz)

FAB-MS (m/z): 700 (M+1)⁺

Compound (H)

¹ H-NMR (CDCl₃) δ (ppm): 1.294(3H, t, J=7.4 Hz), 1.799(3H, s), 2.149(1H,dd, J=5.0, 14.6 Hz), 2.273(3H, s), 2.533(2H, q, J=7.4 Hz), 2.813 (3H,s), 3.972(1H, dd, J=7.4, 14.6 Hz), 4.008(3H, s), 4.015(2H, s), 4.951(2H, s), 5.377(1H, d, J=17.4 Hz), 5.418(1H, d, J=17.4 Hz), 6.973(1H, dd,J=5.0, 7.5 Hz), 7.481-8.037 (5H, m), 9.093(1H, d, J=1.2 Hz)

FAB-MS (m/z): 656 (M+1)⁺

Substantially the same procedure as in Example 20 was repeated using 34mg of Compound (G) to give 18.7 mg of Compound II-51.

¹ H-NMR (CDCl₃) δ (ppm): 1.300(3H, t, J=7.4 Hz), 1.325(3H, t, J=7.4 Hz),2.185(3H, s), 2.514(1H, dd, J=4.8, 14.5 Hz), 2.540(2H, q, J=7.4 Hz),2.555(2H, q, J=7.4 Hz), 3.384(1H, dd, J=7.5, 14.5 Hz), 3.941(2H, s),3.976(2H, s), 4.094(3H, s), 4.836(1H, d, J=16.4 Hz), 4.910(1H, d, J=16.3Hz), 5.781 (1H, s),6.845 (1H, dd, J=4.8, 7.5 Hz), 7.371-7.843(5H,m),8.998(1H, s)

FAB-MS (m/z): 616 (M+1)⁺

Substantially the same procedure as in Example 20 was repeated using 30mg of Compound (H) to give 20.4 mg of Compound II-56.

¹ H-NMR (CDCl₃) δ (ppm): 1.280(3H, t, J=7.4 Hz), 2.144(3H, s), 2.391(1H,dd, J=4.9, 14.5 Hz), 2.517(2H, q,J=7.4 Hz), 3.320(1H, dd, J=7.4, 14.5Hz), 3.885(2H, s), 4.069(3H, s), 4.521(1H, d, J=16.3 Hz), 4.631(1H, d,J=16.7 Hz), 4.804(2H, s), 5.769(1H, s), 6.830(1H, dd, J=4.8, 7.4 Hz),7.375-7.771(5H, m), 8.934(1H, s)

FAB-MS (m/z): 572 (M+1)⁺

EXAMPLE 22

Compound IV-2

Compound II (Z¹, Z² ═H; R¹ ═Br; R² ═H; R═OH; X═CO₂ CH₃) (JapanesePublished Unexamined Patent Application No. 120388/87; herebyincorporated by reference) (50 mg, 0.09 mmol) was dissolved in a mixtureof 0.5 ml of trifluoroacetic acid and 50 μl of 3N HCl, and the solutionwas stirred at room temperature for 2 days. The precipitates werecollected by filtration and subjected to high performance liquidchromatography (Unisil ₅ C₁₈ ; methanol/water=8/2) to give 8.4 mg ofCompound (IV-2).

¹ H-NMR (DMSO-d₆) δ (ppm): 4.947 (2H, s), 7.300-8.010 (6H, m), 8.249(1H,s), 9.266(1H, d, J=2.0 Hz)

FAB-MS (m/z): 390 (M+1)⁺

EXAMPLE 23

Compound II-45 can be prepared by the reaction steps shown in FIG. 23.The starting Compound (J) is disclosed in Japanese Published UnexaminedPatent Application No. 120388/87 (hereby incorporated by reference).

Compound II-45

Compound (J) (200 mg) was dissolved in 1 ml of dimethylformamide, andthen 0.25 ml of an aqueous solution of 23.5 mg of sodium hydroxide wasadded thereto, followed by stirring at room temperature for 4 hours.After 1N hydrochloric acid was added to adjust the pH of the solution to1-2, the precipitates were collected by filtration to give 178 mg (yield91%) of Compound (K).

¹ H-NMR (DMSO-d₆) δ (ppm): 1.965(1H, dd, J=4.8, 14.0 Hz), 2.184(3H, s),3.364(1H, dd, J=7.5, 14.0 Hz), 5.029 (1H, d, J=18.1 Hz), 5.071(1H, d,J=18.0 Hz), 7.133 (1H, dd, J=4.9, 7.5 Hz), 7.595-8.189(5H, m), 8.733(1H, s), 9.398(1H, d, J=2.1 Hz)

Compound (K) (168 mg), was dissolved in 3 ml of pyridine, and then 0.44ml (4.7 mmol) of acetic anhydride was added thereto, followed bystirring at room temperature for 4 days. After evaporation of thesolvent, 4 ml of 1N hydrochloric acid was added to the residue, and theprecipitates were collected by filtration to give 182 mg (yieldquantitative) of Compound (L).

¹ H-NMR (DMSO-d₆) δ (ppm): 1.684(3H, s), 2.135(1H, dd, J=4.9, 14.4 Hz),2.252(3H, s), 3.865(1H, dd, J=7.6, 14.5 Hz), 5.063(2H, s), 7.255(1H, dd,J=4.9, 7.5 Hz), 7.612-8.582(5H, m), 8.760(1H, s), 9.389(1H, d, J=2.1 Hz)

Compound (L) (172 mg) was suspended in thionyl chloride, followed bystirring at 90° C. for 4.5 hours. After evaporation of the solvent,diethyl ether was added to the residue and the precipitates werecollected by filtration to give 180 mg of Compound (M).

Compound (M) (67 mg, 0.1 mmol) was dissolved in 2 ml of ethylenedichloride, and then 180 μl of aniline in tetrahydrofuran was addedthereto under ice cooling, followed by stirring at the same temperaturefor 1 hour. After evaporation of the solvent, the residue was dissolvedin a mixture of 2 ml of tetrahydrofuran and 0.5 ml of methanol, and then1 ml of 1N NaOH was added thereto, followed by stirring at roomtemperature for 3 hours. To the solution was added 1N hydrochloric acid(1.2 ml) for neutralization, followed by dilution with tetrahydrofuran.The mixture was washed with a saline solution and dried over sodiumsulfate. After evaporation of the solvent, the residue was subjected tosilica gel column chromatography (chloroform/methanol=98/2) to giveCompound II-45 (13 mg from 56 mg of isolated Compound N).

¹ H-NMR (DMSO-d₆) δ (ppm): 2.110(1H, dd, J=4.9, 13.9 Hz), 2.175(3H, s),5.019(1H, d, J=18.1 Hz), 5.088(1H, d, J=18.0 Hz), 6.887(1H, s),7.119-8.201(11H, m), 8.711 (1H, s), 9.391(1H, d, J=2.2 Hz), 10.071(1H,s)

FAB-MS (m/z): 687 (M+1)⁺

EXAMPLE 24 ##STR68##

The starting compound (Q) is disclosed in Japanese Unexamined PatentApplication No. 295588/88.

Compound (Q) (50 mg, 0.0861 mmol) was dissolved in 3 ml of chloroform,and then 200 mg (1.41 mmol) of 2-dimethylaminoethanethiol hydrochlorideand 49 mg (0.21 mmol) of (±)-10-camphorsulfonic acid were added thereto,followed by stirring at room temperature for 12 hours. The reactionmixture was washed successively with a saturated aqueous solution ofsodium bicarbonate, water, and a saline solution, and dried over sodiumsulfate. After evaporation of the solvent, the residue was subjected topreparative thin layer chromatography (chloroform/methanol=99/1) to give56.3 mg (yield 98%) of N,O-diacetylated Compound II-65.

FAB-MS (m/z): 668 (M+1)⁺

N,O-diacetylated Compound II-65 (36.6 mg, 0.0548 mmol) was dissolved ina mixture of 6 ml of chloroform and 3 ml of methanol, and then 18 μl(0.09 mmol) of 5.1N sodium methoxide was added thereto, followed bystirring at room temperature for 20 minutes. Amberlyst® 15 ion-exchangeresin (100 mg) was added to the reaction mixture, followed by stirringfor one hour, and insoluble material was separated by filtration. Afterevaporation of the solvent, the residue was subjected to preparativethin layer chromatography (chloroform/methanol=97/3) to give 28.4 mg(yield 89%) of Compound II-65.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.011 (1H, dd, J=4.9, 14.1 Hz), 2.142 (9H,s), 2.460-2.584 (4H, m), 3.404 (1H, dd, J=7.3, 14.1 Hz), 3.923 (3H, s),3.950 (2H, s), 4.951-5.054 (2H, m), 6.336 (1H, s), 7.111 (1H, dd, J=4.9,7.3 Hz), 7.338-8.060 (6H, m), 8.595 (1H, s), 9.137 (1H, d, J=1.3 Hz)

FAB-MS (m/z): 585 (M+1)⁺

EXAMPLE 25

Compound II-66

Compound II-66 is prepared, e.g., according to a method of Japanesepublished unexamined Patent Application No. 155284/87 (herebyincorporated by reference).

EXAMPLE 26

Compound II-75

Compound (P) (Japanese Published Unexamined Patent Application No.295588/88) (100 mg, 0.173 mmol) and 4-amino-1,2,4-triazole (17.4 mg,0.207 mmol) were dissolved in a mixture of 4 ml of chloroform and 1.5 mlof tetrahydrofuran, and then 0.05 ml of 3N hydrochloric acid was addedthereto, followed by stirring at room temperature for 3.5 hours. Afterethyl acetate was added thereto, insoluble matters were collected byfiltration and subjected to silica gel column chromatography(chloroform/methanol=95/5) to give 71.9 mg (yield 64%) ofN,O-diacetylated Compound II-75.

FABS-MS (m/z): 646 (M+1)⁺

N,O-Diacetylated Compound II-75 (37.5 mg, 0.058 mmol) was dissolved in amixture of 2 ml of 1,2-dichloroethane and 0.6 ml of methanol, and then11 μl (0.058 mmol) of 5.1N sodium methoxide in methanol was addedthereto, followed by stirring at room temperature for 20 minutes.Amberlyst® 15 (50 mg) was added to the reaction mixture, followed bystirring for 30 minutes, and insoluble matters were filtered off. Theinsoluble matters were washed well withdichloromethane/methanol/ammonium hydroxide (8/2/0.5), and the combinedfiltrate was concentrated under reduced pressure. The residue wassubjected to silica gel column chromatography to give 26.8 mg (yield82%) of compound II-75.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.105 (1H, dd, J=5.0, 14.1 Hz), 2.157 (3H,s), 3.444 (1H, dd, J=7.5, 14.1 Hz), 3.973 (3H, s), 5.020 (1H, d, J=17.2Hz), 5.076 (1H, d, J=17.2 Hz), 6.399 (1H, s), 7.226 (1H, dd, J=5.0, 7.5Hz), 7.366-8.114 (6H, m), 8.708 (1H, s), 9.219 (2H, s), 9.260 (1H, s),9.701 (1H, d, J=1.5 Hz)

FAB-MS (m/z): 562 (M+1)⁺

EXAMPLE 27

Compound II-79

Compound (Q) (Japanese Published Unexamined Patent Application No.295588/88 (50 mg, 0.0861 mmol) and 2-(butylamino)ethanethiol (0.127 ml,0.861 mmol) were dissolved in chloroform, and then 300 mg (1.29 mmol) ofcamphorsulfonic acid was added thereto, followed by stirring at roomtemperature for 4 days. A saturated aqueous solution of sodiumbicarbonate was added to the reaction mixture, and the organic layer waswashed with an aqueous solution of sodium chloride and dried overmagnesium sulfate. The solvent was evaporated under reduced pressure,and the residue was subjected to silica gel column chromatography(chloroform/methanol=95/5) to give 34.6 mg (yield 58%) ofN,O-diacetylated Compound II-79.

FAB -MS (m/z): 697 (M+1)⁺

Substantially the same procedure as in Example 26 was repeated using31.1 mg (0.0447 mmol) of N,O-diacetylated Compound II-79 to giveCompound II-79 (yield 52%).

¹ H-NMR (DMSO-d₆) δ (ppm): 0.855 (3H, t, J=7.4 Hz), 1.286 (2H, m), 1.510(2H, m), 2.007 (1H, dd, J=4.9, 14.1 Hz), 2.148 (3H, s), 2.731 (2H, m),2.843 (2H, m), 3.106 (2H, m), 3.389 (1H, dd, J=7.4, 14.1 Hz), 3.927 (3H,s), 4.032 (2H, s), 4.987 (1H, d, J=17.6 Hz), 5.030 (1H, d, J=17.6 Hz),6.345 (1H, s), 7.126 (1H, dd, J=4.9, 7.4 Hz), 7.350-8.067 (6H, m), 8.614(1H, s), 9.161 (1H, s)

FAB-MS (m/z): 613 (M+1)⁺

EXAMPLE 28

Compound II-80

Compound F (W094/02488, hereby incorporated by reference) (6.19 g, 10.1mmol) was dissolved in a mixture of 300 ml of 1,2-dichloroethane and 100ml of methanol, and then 0.5 ml (2.55 mmol) of 5.1N sodium methoxide inmethanol was added thereto, followed by stirring at room temperature for35 minutes. The reaction mixture was poured into ice-water, andinsoluble matters were collected by filtration to give 4.95 g (yield93%) of a compound having bis(hydroxymethyl) in place ofbis(dimethylaminoethylthiomethyl) of Compound II-80.

FAB-MS (m/z): 528 (M+1)⁺

Substantially the same procedure as in Example 27 was repeated using22.1 mg (0.0419 mmol) of the compound having bis(hydroxymethyl) in placeof bis(dimethylaminoethylthiomethyl) of Compound II-80 and 59.4 mg(0.419 mmol) of 2-(dimethylamino) ethanethiol hydrochloride to give 13.1mg (yield 45%) of Compound II-80.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.999 (1H, dd, J=4.9, 14.2 Hz), 2.134 (3H,s), 2.143 (6H, s), 2.149 (6H, s), 2.461-2.585 (8H, m), 3.378 (1H, dd,J=7.3, 14.2 Hz), 3.922 (3H, s), 3.950 (2H, s), 3.983 (2H, s), 4.954 (1H,d, J=17.7 Hz), 5.012 (1H, d, J=17.7 Hz), 6.322 (1H, s), 7.108 (1H, dd,J=4.9, 7.3 Hz), 7.444-7.952 (4H, m), 8.616 (1H, s), 9.133 (1H, d, J=1.4Hz)

FAB-MS (m/z): 702 (M+1)⁺

EXAMPLE 29

Compound II-72

Substantially the same procedure as in Example 27 was repeated using 50mg (0.0861 mmol) of Compound (Q) and 97.8 mg (0.861 mmol) of2-aminoethanethiol hydrochloride to give 49.6 mg (yield 90%) ofN,O-diacetylated Compound II-72.

FAB-MS (m/z): 641 (M+1)⁺

Substantially the same procedure as in Example 26 was repeated using39.5 mg (0.0617 mmol) of N,O-diacetylated Compound II-72 to give 30.2 mg(yield 88%) of Compound II-72.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.014 (1H, dd, J=4.9, 14.1 Hz), 2.146 (3H,s), 2.519 (2H, t, J=7.2 Hz), 2.748 (2H, t, J=7.2 Hz), 3.386 (1H, dd,J=7.5, 14.1 Hz), 3.925 (3H, s), 3,936 (2H, s), 4.979 (1H, d, J=17.0 Hz),5.029 (1H, d, J=17.0 Hz), 6.330 (1H, s), 7.111 (1H, dd, J=4.9, 7.5 Hz),7.344-8.059 (6H, m), 8.600 (1H, s), 9.131 (1H, d, J=1.5 Hz)

FAB-MS (m/z): 557 (M+1)⁺

EXAMPLE 30

Compound VI-1

Compound (R) (I. Antibiotics, 38:1437, 1985, FIG. 24) (1 g, 1.81 mmol)was dissolved in 50 ml of 1,2-dichloroethane, and then 0.17 ml (3.80mmol) of fuming nitric acid was added dropwise thereto at 0° C.,followed by stirring at room temperature for 20 minutes. After thereaction mixture was diluted with chloroform, a saturated aqueoussolution of sodium bicarbonate was added thereto, and the organic layerwas washed with an aqueous solution of sodium chloride and dried overmagnesium sulfate. After evaporation of the solvent under reducedpressure, 40 ml of dimethylformamide and 600 mg of 10% Pd/C were addedto the residue, followed by stirring at 60° C. for one hour in anatmosphere of hydrogen. Insoluble material was filtered off, and thefiltrate was concentrated under reduced pressure. The residue wassubjected to silica gel column chromatography (ethylacetate/toluene=20/80) to give 130.8 mg (yield 13%) of an aminederivative.

FAB-MS (m/z): 567 (M+1)⁺

The amine derivative (23.9 mg, 0.0422 mmol) was dissolved in 2 ml ofchloroform, and then 9.2 μl (0.0660 mmol) of triethylamine and 87 μl(1.10 mmol) of ethyl isocyanate were added thereto, followed by stirringat room temperature for 2 days. Water, methanol, and chloroform wereadded to the reaction mixture to complete the reaction, and the mixturewas extracted with chloroform. The organic layer was washed with anaqueous solution of sodium chloride and dried over sodium sulfate. Afterevaporation of the solvent under reduced pressure, the residue wassubjected to silica gel column chromatography (chloroform/methanol=98/2)to give 21.4 mg (yield 80%) of N,O-diacetylated Compound VI-1.

Substantially the same procedure as in Example 26 was repeated using21.4 mg (0.0336 mmol) of N,O-diacetylated Compound VI-1 to give 17.0 mg(yield 91%) of Compound VI-1.

1H-NMR (DMSO-d₆) δ (ppm): 1.129 (3H, t, J=7.1 Hz), 2.086 (3H, s), 2.110(1H, dd, J=5.5, 14.3 Hz), 3.180 (2H, m), 3.237 (1H, dd, J=7.4, 14.3 Hz),3.892 (3H, s), 4.984 (1H, d, J=17.0 Hz), 5.030 (1H, d, J=17.0 Hz), 6.359(1H, s), 6.457 (1H, t, J=5.7 Hz), 7.157-7.230 (2H, m), 7.272 (1H, dd,J=5.5, 7.4 Hz), 7.344-8.058 (4H, m), 8.185 (1H, s), 8.616 (1H, s), 9.243(1H, dd, J=1.3, 7.8 Hz)

FAB-MS (m/z): 554 (M+1)¹

EXAMPLE 31

Compound VI-2

Substantially the same procedure as in Example 30 was repeated using 5 g(9.07 mmol) of Compound (R; FIG. 24) to give 259 mg (yield 5%) of adiamine derivative.

FAB-MS (m/z): 582 (M+1)⁺

Substantially the same procedure as in Example 26 was repeated using24.5 mg (0.0422 mmol) of the diamine derivative to give 3.8 mg (yield18%) of Compound VI-2.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.952 (1H, dd, J=5.4, 13.9 Hz), 2.062 (3H,s), 3.894 (3H, s), 4.818-5.339 (6H, m), 6.198 (1H, s), 6.826-7.207 (4H,m), 7.507 (1H, dd, J=5.4, 7.3 Hz), 7.630 (1H, d, J=8.8 Hz), 8.443 (1H,s), 8.770 (1H, dd, J=1.2, 7.8 Hz)

FAB-MS (m/z): 498 (M+1)⁺

EXAMPLE 32

Compound IV-6

Compound (S; FIG. 25) J. Chem. Soc. Perkin. Trans. 1, 2475 (1990)! (5.15g, 13.0 mmol) was dissolved in a mixture of 30 ml of dimethylformamideand 60 ml of toluene, and then 1.45 g (12.9 mmol) of potassiumtert-butoxide was added thereto at -20° C. in a atmosphere of argon,followed by stirring at room temperature for 30 minutes. After coolingthe reaction mixture to -20° C., 1.12 ml (12.9 mmol) of allyl bromidewas added thereto and the mixture was stirred at 0° C. for 2 hours. Thesolvent was evaporated under reduced pressure, and water was added tothe residue, followed by extraction with tetrahydrofuran. The organiclayer was washed with an aqueous solution of sodium chloride, and driedover magnesium sulfate. After evaporation of the solvent, the residuewas subjected to silica gel column chromatography (ethylacetate/toluene=1/15), and triturated with dichloromethane to give 898.4mg (yield 16%) of Compound (T-1) as a single regioisomer.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.56-1.61 (2H, m), 1.73-1.87 (2H, m),2.00-2.14 (2H, m), 3.63-3.69 (1H, m), 3.99-4.02 (1H, m), 4.747 (1H, dd,J=1.5, 17.1 Hz), 5.053 (1H, dd, J=1.5, 10.4 Hz), 5.084 (1H, d, J=17.3Hz), 5.138 (1H, d, J=17.3Hz), 5.462 (1H, dd, J=2.0, 11.0 Hz), 5.593 (2H,d, J=4.6Hz), 6.178 (1H, ddt, J=4.6, 10.4 17.1 Hz), 7.242 (1H, ddd,J=0.9, 7.0,7.9 Hz), 7.368 (1H, dd, J=7.2, 7.8 Hz), 7.455 (1H, ddd,J=1.2, 7.0, 8.2 Hz), 7.542 (1H, ddd, J=1.1, 7.2, 8.3 Hz), 7.711 (1H, dd,J=0.9, 8.2 Hz), 7.762 (1H, d, J=8.3 Hz), 8.177 (1H, d, J=7.8 Hz), 9.305(1H, d, J=7.9 Hz), 11.573 (1H, s)

FAB-MS (m/z): 436 (M+1)⁺

Compound (T-1) (1.44 g, 3.30 mmol) was dissolved in 50 ml oftetrahydrofuran, and then 4.05 g (33.2 mmol) of 9-borabicyclo3,3,1!nonane (9-BBN) (dimer) was added thereto, followed by stirring atroom temperature for 3 hours in an atmosphere of argon. After coolingthe reaction mixture to 0° C., 6 ml of 1N sodium hydroxide and 6 ml of a35% aqueous solution of hydrogen peroxide were added thereto, followedby stirring for 45 minutes. After dilution of he reaction mixture withwater, the mixture was extracted with ethyl acetate. The organic layerwas washed successively with water and an aqueous solution of sodiumchloride, and dried over magnesium sulfate. The solvent was evaporatedunder reduced pressure, and the residue was subjected to silica gelcolumn chromatography (chloroform/methanol=100/1) to give 875.5 mg(yield 58%) of Compound (J-1).

¹ H-NMR (DMSO-d₆) δ (ppm): 1.5-1.6 (2H, brm), 1.7-1.9 (2H, brm), 2.0-2.2(2H, brm), 2.08-2.14 (2H, m), 3.49-3.53 (2H, m), 3.62-3.68 (1H, m),3.99-4.02 (1H, m), 4.962 (2H, t, J=6.9 Hz), 5.072 (1H, d, J=17.2 Hz),5.081 (1H, t, J=4.7 Hz), 5.123 (1H, d, J=17.2 Hz), 5.458 (1H, dd, J=2.0.11.0 Hz), 7.251 (1H, ddd, J=0.9,7.0, 7.9 Hz)7.358 (1H, dd, J=7.2, 7.8Hz), 7.463 (1H, ddd, J=1.2, 7.0, 8.2 Hz), 7.555 (1H, ddd, J=1.1, 7.2,8.3 Hz), 7.696 (1H, d, J=8.2 Hz), 7.825 (1H, d, J=8.3 Hz), 8.162 (1H, d,J=7.8 Hz), 9.311 (1H, d, J=7.9 Hz), 11.684 (1H, s)

FAB-MS (m/z): 454 (M+1)⁺

Compound (U-1) (178.5 mg, 0.394 mmol) was dissolved in 10 ml ofdimethylformamide, and then 309.5 mg (1.18 mmol) of triphenylphosphineand 0.060 ml (1.2 mmol) of bromine were added thereto at 0° C. under anatmosphere of argon, followed by stirring at room temperature for 3hours. After water was added to the reaction mixture to complete thereaction, the mixture was extracted with ethyl acetate, and the organiclayer was washed successively with water and an aqueous solution ofsodium chloride, and dried over magnesium sulfate. After evaporation ofthe solvent, the residue was subjected to silica gel columnchromatography (ethyl acetate/toluene=1/8) to give 134.6 mg (yield 66%)of Compound (W).

¹ H-NMR (CDCl₃) δ (ppm): 1.68-2.10 (6H, m), 2.13-2.18 (2H, m), 3.542(2H, t, J=5.7 Hz), 3.80-3.86 (1H, m), 4.14-4.20 (1H, m), 4.658 (2H, t,J=7.5 Hz), 4.674 (1H, d,J=16.6 Hz), 4.830 (1H, d, J=16.6 Hz), 5.611 (1H,dd, J=2.5, 10.6 Hz), 7.13-7.52 (6H, m), 7.746 (1H, d J=7.6 Hz), 8.884(1H, s), 9.294 (1H, d, J=8.0 Hz)

FAB-MS (m/z): 516 (M+1)⁺

Compound (W) was dissolved in 5 ml of dimethylformamide, and then 0.045ml (0.52 mmol) of morpholine was added thereto, followed by stirring at80° C. for 3 hours in an atmosphere of argon. After cooling the reactionmixture to room temperature, ice-water was added thereto, and the formedprecipitates were collected by filtration. The precipitates were driedunder reduced pressure, and subjected to thin layer chromatography(chloroform/methanol=25/1). The product obtained was dissolved in 10 mlof tetrahydrofuran, and then 8 ml of 4N sulfuric acid was added thereto,followed by stirring at 60° C. for 12 hours. After cooling the reactionmixture to room temperature, ice was added thereto, and the mixture wasextracted with ethyl acetate. The organic layer was washed successivelywith water and an aqueous solution of sodium chloride, and dried overmagnesium sulfate. The solvent was evaporated under reduced pressure,and the residue was subjected to silica gel column chromatography (ethylacetate/toluene=1/2). The product obtained was dissolved in a mixture ofchloroform and ethyl acetate, and then 0.88N hydrochloric acid in ethylacetate was added, followed by stirring at room temperature for onehour. The formed precipitates were collected by filtration, washed withethyl acetate, and dried under reduced pressure to give 35.0 mg (yield19%) of Compound (IV-6).

¹ H-NMR (DMSO-d₆) δ (ppm): 2.29-2.34 (2H, m), 2.96-3.04 (2H, m),3.30-3.40 (4H, m), 3.66-3.72 (2H, m), 3.56-3.90 (2H, m), 4.972 (2H, s),5.093 (2H, t, J=7.1 Hz), 7.245 (1H, ddd, J=0.9, 7.0, 7.9 Hz), 7.370 (1H,dd, J=7.0, 7.9 Hz), 7.458 (1H, ddd, J=1.2, 7.0, 8.2 Hz), 7.565 (1H, ddd,J=1.2, 7.0, 8.2 Hz), 7.799 (1H, d, J=8.2 Hz), 7.884 (1H, d, J=8.2Hz)8.071 (1H, d, J=7.9 Hz), 8.516 (1H, s), 9.345 (1H, d, J=7.9 Hz),10.4-10.6 (1H, brs), 11.823 (1H, s)

FAB-MS (m/z): 439 (M+1)⁺

EXAMPLE 33 ##STR69##

Compound (S) (J. Chem. Soc. Perkin Trans. 1:2475 1990) (823.7 mg, 2.083mmol) was dissolved in 20 ml of dimethylformamide, and 166.4 mg (4.16mmol) of sodium hydride (60%) was added thereto under ice cooling,followed by stirring at the same temperature for 10 minutes. Allylbromide (0.45 ml, 5.2 mmol) was added thereto and the solution wasstirred for 2 hours under ice cooling. After dilution with chloroform,water was added thereto and the organic layer was separated, washed witha saline solution, and dried over magnesium sulfate. After evaporationof the solvent, the residue was subjected to silica gel columnchromatography (ethyl acetate/toluene=1/15) to give 735.0 mg (yield 74%)of Compound (T-2).

¹ H-NMR (DMSO-d₆) δ (ppm): 1.563-2.154(6H, m), 3.657(1H, m), 4.008(1H,m), 5.044-5.478(11H, m), 6.153(2H, m), 7.240-7.640(6H, m), 8.167(1H, d,J=7.8 Hz), 9.415(1H, d, J=7.8 Hz)

FAB-MS (m/z): 476 (M+1)⁺

Sodium borohydride (77.7 mg, 2.05 mmol) was suspended in 20 ml oftetrahydrofuran, and 231.0 mg (1.82 mmol) of iodine was added thereto at0° C. in an atmosphere of argon, followed by stirring at the sametemperature for 15 minutes. Compound (T-2) (136.7 mg, 0.287 mmol) wasadded thereto at the same temperature and the mixture was stirred atroom temperature for 4.5 hours. After the reaction mixture was cooled to0° C., 3.7 ml of 1N sodium hydroxide and 3.7 ml of a 35% aqueoussolution of hydrogen peroxide were added thereto, followed by stirringfor a further 30 minutes. The reaction mixture was diluted with waterand extracted with ethyl acetate. The ethyl acetate layer was washedsuccessively with water and a saline solution, and dried over magnesiumsulfate. After evaporation of the solvent, the residue was subjected tosilica gel column chromatography (chloroform/methanol=15/1) to give 88.9mg (yield 61%) of Compound (U-2).

¹ H-NMR (CDCl₃) δ (ppm): 1.60-2.11(10H, m), 3.129(2H, t, J=5.9 Hz),3.192(2H, t, J=5.9 Hz), 3.798(1H, dt, J=2.8, 11.7 Hz), 4.09-4.15(1H, m),4.723(2H, t, J=7.2 Hz), 4.807(2H, t, J=7.2 Hz), 4.943(1H, d, J=16.6 Hz),5.107(1H, d, J=16.6 Hz), 5.652(1H, dd, J=2.4, 10.5 Hz), 7.15-7.18(1H,m), 7.318(1H, ddd, J=1.1, 7.0, 8.0 Hz), 7.35-7.39(1H, m), 7.461(1H, ddd,J=1.2, 6.8, 8.0 Hz), 7.519(1H, dd, J=1.0, 8.0 Hz), 7.610(1H, d, J=8.0Hz), 7.951(1H, d, J=8.0 Hz), 9.490(1H, d, J=8.0 Hz)

FAB-MS (m/z): 512 (M+1)⁺

Compound (U-2) (88.9 mg, 0.174 mmol) was dissolved in 10 ml oftetrahydrofuran, and 8 ml of 4N sulfuric acid was added thereto,followed by stirring at 60° C. for 24 hours. After the reaction mixturewas cooled to room temperature, ice was added thereto, followed byextraction with ethyl acetate. The ethyl acetate layer was washedsuccessively with water and a saline solution, and dried over magnesiumsulfate. After evaporation of the solvent, the residue was subjected tothin layer chromatography (chloroform/methanol=15/1) to give 37.6 mg(yield 51%) of Compound IV-5.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.59-1.65(2H, m), 1.70-1.82(2H, m),3.03-3.27(2H, m), 3.09-3.14(2H, m), 4.371(1H, t, J=5.0 Hz), 4.419(1H, t,J=5.0 Hz), 4.780(2H, t, J=7.3 Hz), 4.818(2H, t, J=7.4 Hz), 4.972(2H, s),7.288(1H, ddd, J=0.8, 7.0, 7.8 Hz), 7.370(1H, t, J=7.2 Hz), 7.501(1H,ddd, J=1.2, 7.0, 8.2 Hz), 7.563(1H, ddd, J=1.1, 7.2, 8.3 Hz), 7.779(1H,d, J=8.3 Hz), 7.848(1H, d, J=8.2 Hz), 8.043(1H, d, J=7.2 Hz), 9.412(1H,dd, J=0.8, 7.8 Hz)

FAB-MS (m/z): 428 (M+1)⁺

EXAMPLE 34

Compound II-68

Compound (Q) (50.1 mg, 0.0862 mmol) was dissolved in 3 ml of chloroform,and then 129.5 mg (0.862 mmol) of 2-mercaptobenzimidazole and 49 mg(0.21 mmol) of (±)-10-camphorsulfonic acid were added thereto, followedby stirring at room temperature for 12 hours. The reaction mixture waswashed successively with a saturated aqueous solution of sodiumbicarbonate, water, and a saline solution, and dried over sodiumsulfate. After evaporation of the solvent, the residue was subjected topreparative thin layer chromatography (chloroform/methanol=99/1) to give46 mg (yield 75%) of N,O-diacetylated Compound II-68.

FAB-MS (m/z): 714 (M+1)⁺

Substantially the same procedure as in Example 25 was repeated using33.4 mg (0.0468 mmol) of N,O-diacetylated Compound II-68 to give 17.5 mg(yield 59%) of Compound II-68.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.995 (1H, dd, J=4.9, 14.1 Hz), 2.139 (3H,s), 3.914 (3H, s), 4.799 (2H, s), 4.979 (1H, d, J=17.3 Hz), 5.028 (1H,d, J=17.3 Hz), 6.342 (1H, s), 7.101 (1H, dd, J=4.9, 7.3 Hz), 7.123-8.056(10H, m), 8.617 (1H, s), 9.278 (1H, m)

FAB-MS (m/z): 630 (M+1)⁺

EXAMPLE 35

Compound II-69

Substantially the same procedure as in Example 25 was followed using 50mg (0.0861 mmol) of Compound Q and 0.0868 ml (0.861 mmol) offurfurylmercaptan to give 36.0 mg (yield 62%) of N,O-diacetylatedCompound II-69.

FAB-MS (m/z): 678 (M+1)⁺

Substantially the same procedure as in Example 25 was repeated using22.7 mg (0.0335 mmol) of N,O-diacetylated Compound II-69 to give 17.7 mg(yield 89%) of Compound II-69.

¹ H-NMR (CDCl₃) δ (ppm): 2.209(3H, s)2.607(1H, dd, J=4.9, 14.5 Hz),3.401(1H, dd, J=7.5, 14.5 Hz), 3.671(2H, s), 3.857(2H, s), 4.103(3H, s),4.532(1H, brs), 4.789(1H, d, J=16.1 Hz), 4.873(1H, d, J=16.1 Hz),5.690(1H, s), 6.378(1H, dd, J=1.9, 3.2 Hz), 6.416(1H, dd, J=0.6, 3.2Hz), 6.846(1H, dd, J=4.8, 7.5 Hz), 7.334-7.932(7H, m), 8.961(1H, m)

FAB-MS (m/z): 593 (M)⁺

EXAMPLE 36

Compound II-70

Compound (P) (100 mg, 0.173 mmol) was dissolved in 4 ml of chloroform,and then 34.0 mg (0.277 mmol) of 1-aminopyrrolidine hydrochloride wasadded thereto, followed by stirring at room temperature for 4 hours.After evaporation of the solvent under reduced pressure, the residue wassubjected to silica gel column chromatography (chloroform/methanol=99/1)to give 100.5 mg (yield 90%) of N,O-diacetylated Compound II-70.

FAB-MS (m/z): 648 (M+1)⁺

Substantially the same procedure as in Example 25 was repeated using 40mg (0.0618 mmol) of N,O-diacetylated Compound II-70 to give 30 mg (yield86%) of Compound II-70.

H-NMR (DMSO-d₆ δ (ppm): 1.910-1.937(4H, m), 2.031(1H, dd, J=4.9, 14.1Hz), 2.142(3H, s), 2.329-2.635(4H, m), 3.395(1H, dd, J=7.3, 14.1 Hz),3.925(3H, s), 4.981(1H, d, J=17.0 Hz), 5.030(1H, d, J=17.0 Hz),7.110(1H, dd, J=4.9, 7.3 Hz), 7.345-8.057(6H, m), 7.425(1H, s),8.596(1H, s), 9.210(1H, d, J=1.4 Hz)

FAB-MS (m/z): 564 (M+1)⁺

EXAMPLE 37

Compound II-71

Compound (P) (49 mg, 0.0846 mmol) was dissolved in 3 ml of chloroform,and then a solution of 15.8 mg (0.145 mmol) of 2-hydrazinopyridine inchloroform and 49 mg (0.21 mmol) of (±)-10-camphorsulfonic acid wereadded thereto, followed by stirring at room temperature for 12 hours.The reaction mixture was washed successively with a saturated aqueoussolution of sodium bicarbonate, water, and a saline solution, and driedover sodium sulfate. After evaporation of the solvent, the residue wassubjected to preparative thin layer chromatography(chloroform/methanol=99/1) to give 35.8 mg (yield 64%) ofN,O-diacetylated Compound II-71.

FAB-MS (m/z): 671 (M+1)⁺

Substantially the same procedure as in Example 25 was repeated using24.6 mg (0.0367 mmol) of N,O-diacetylated Compound II-71 to give 11.8 mg(yield 55%) of Compound II-71.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.039 (1H, dd, J=5.0, 13.9 Hz), 2.153 (3H,s), 3.418 (1H, dd, J=7.2, 13.9 Hz), 3.933 (3H, s), 5.001 (1H, d, J=17.5Hz), 5.057 (1H, d, J=17.5 Hz), 6.366 (1H, s), 6.748 (1H, m), 7.164 (1H,dd, J=5.0, 7.2 Hz), 7.301-8.120 (9H, m), 8.242 (1H, s), 8.656 (1H, s),8.656 (1H, s), 9.368 (1H, s), 10.738 (1H, s)

FAB-MS (m/z): 587 (M+1)⁺

EXAMPLE 38

Compound II-73

Substantially the same procedure as in Example 25 was followed using 30mg (0.0516 mmol) of Compound (Q) and 52.2 mg (0.516 mmol) of1H-1,2,4-triazole-3-thiol to give 31.4 mg (yield 92%) ofN,O-diacetylated Compound II-73.

FAB-MS (m/z): 665 (M+1)⁺

Substantially the same procedure as in Example 25 was repeated using 15mg (0.0226 mmol) of N,O-diacetylated Compound II-73 to give crudeCompound II-73. Chloroform/methanol (90/10) was added thereto, followedby stirring to give 10.9 mg (yield 83%) of Compound II-73 as aprecipitate.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.006(1H, dd, J=4.9, 13.9 Hz), 2.144(3H, s),3.375(1H, dd, J=7.3, 13.9 Hz), 3.921 (3H, s), 4.559(2H, brs), 4.977(1H,d, J=17.4 Hz), 5.033(1H, d, J=17.4 Hz), 6.332(1H, s), 7.106(1H, dd,J=4.9, 7.3 Hz), 7.341-8.062(6H, m), 8.614(1H, s), 9.202(1H, d, J=1.5 Hz)

FAB-MS (m/z): 581 (M+1)⁺

EXAMPLE 39

Compound II-74

Compound (P) (97.5 mg, 0.168 mmol) was dissolved in 4 ml oftetrahydrofuran, and then an aqueous solution of 25.1 mg (0.0950 mmol)of aminoguanidine sulfate was added thereto, followed by stirring atroom temperature for 3 hours. Ethyl acetate was added thereto, followedby stirring, and the insoluble matters were collected by filtration andsubjected to silica gel column chromatography(chloroform/methanol=85/15) to give 87.1 mg (yield 82%) ofN,O-diacetylated Compound II-74.

FAB-MS (m/z): 636 (M+1)⁺

Substantially the same procedure as in Example 25 was repeated using69.6 mg (0.110 mmol) of N,O-diacetylated Compound II-74 to give 37.2 mg(yield 62%) of Compound II-74.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.046(1H, dd, J=4.9, 14.2 Hz), 2.148(3H, s),3.406(1H, dd, J=7.5, 14.2 Hz), 3.929 (3H, s), 4.988(1H, d, J=17.3 Hz),5.045(1H, d, J=17.3 Hz), 5.637-6.129(4H, m), 6.350(1H, s), 7.156(1H, dd,J=4.9, 7.5 Hz), 7.345-8.092(6H, m), 8.206 (1H, s), 8.603(1H, s),9.271(1H, d, J=1.7 Hz)

FAB-MS (m/z): 552(M+1)⁺

EXAMPLE 40

Compound II-76

Compound (P) (103.8 mg, 0.179 mmol) was dissolved in a mixture of 6 mlof chloroform and 3 ml of methanol, and then 0.5 ml of an aqueoussolution of 0.020 ml (0.207 mmol) of 4-aminomorpholine and 0.05 ml of 3Nhydrochloric acid were added thereto, followed by stirring at roomtemperature for 3 hours. The reaction mixture was washed successivelywith a saturated aqueous solution of sodium bicarbonate and a salinesolution, and dried over sodium sulfate. After evaporation of thesolvent, the residue was subjected to silica gel column chromatography(chloroform/methanol =90/100) to give 82.8 mg (yield 70%) ofN,O-diacetylated Compound II-76.

FAB-MS (m/z): 663 (M+1)⁺

Substantially the same procedure as in Example 25 was repeated using50.6 mg (0.0763 mmol) of N,O-diacetylated Compound II-76 to give 36.4 mg(yield 82%) of Compound II-76.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.042(1H, dd, J=4.8, 14.3 Hz), 2.144(3H, s),3.139-3.163(4H, m), 3.404(1H, dd, J=7.5, 14.3 Hz), 3.792-3.815(4H, m),3.927(3H, s), 4.984(1H, d, J=17.3 Hz), 5.040(1H, d, J=17.3 Hz),6.352(1H, s), 7.132(1H, dd, J=4.8, 7.5 Hz), 7.344-8.065(6H, m),7.897(1H, s), 8.610(1H, s), 9.316(1H, d, J=1.7 Hz)

FAB-MS (m/z): 580 (M+1)⁺

EXAMPLE 41

Compound II-77

Substantially the same procedure as in Example 40 was followed using 100mg (0.173 mmol) of Compound P and 16.7 mg (0.173 mmol) of1,1-dimethylhydrazine hydrochloride to give 52.3 mg (yield 49%) ofN,O-diacetylated Compound II-77.

FAB-MS (m/z): 622 (M+1)⁺

Substantially the same procedure as in Example 25 was repeated using38.4 mg (0.0618 mmol) of N,O-diacetylated Compound I-75 to give 10.9 mg(yield 33%) of Compound I-75.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.037(1H, dd, J=5.0, 14.1 Hz), 2.142(3H, s),2.939(6H, s), 3.399(1H, dd, J=7.5, 14.1 Hz), 3.926(3H, s), 4.981(1H, d,J=17.7 Hz), 5.037(1H, d, J=17.7 Hz), 6.342(1H, s), 7.118(1H, dd, J=5.0,7.5 Hz), 7.342-8.063(6H, m), 7.533(1H, s), 8.601(1H, s), 9.258(1H, s)

FAB-MS (m/z): 538 (M+1)⁺

EXAMPLE 42

Compound II-78

Substantially the same procedure as in Example 40 was followed using99.5 mg (0.172 mmol) of Compound (P) and 42.4 mg of1-amino-4-methylpiperazine to give N,O-diacetylated Compound II-78.

Then, substantially the same procedure as in Example 25 was repeatedusing the above N,O-diacetylated Compound II-78 to give 19.4 mg yieldfrom Compound (P)19%! of Compound II-78.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.040(1H, dd, J=5.0 , 14.0 Hz), 2.144(3H, s),2.268(3H, s), 2.553(4H, m), 3.167(4H, m), 3.401(1H, dd, J=7.2, 14.0 Hz),3.927(3H, s) , 4.982(1H, d, J=17.1 Hz), 5.038(1H, d, J=17.1 Hz),6.345(1H, s), 7.128(1H, dd, J=5.0, 7.2 Hz), 7.343-8.065(6H, m),7.827(1H, s), 8.609(1H, s), 9.299(1H, d, J=1.2 Hz)

FAB-MS (m/z) : 593 (M+1)⁺

EXAMPLE 43

Compound II-81

Compound (AA), a compound having bis(hydroxymethyl) in place ofbis(dimethylaminoethylthiomethyl) of Compound II-80 (described inExample 28) (53.9 mg, 0.102 mmol) was dissolved in 2.5 ml ofdichloromethane. Then, 0.18 ml (2.0 mmol) of 2-propanethiol and 0.03 ml(0.2 mmol) of trifluoroacetic anhydride were successively added thereto,followed by stirring at room temperature in an argon stream for 3 hours.A saturated aqueous solution of sodium bicarbonate was added to thereaction mixture, and the mixture was extracted with chloroform. Theorganic layer was washed with an aqueous solution of sodium chloride anddried over sodium sulfate. The solvent was evaporated under reducedpressure, and the residue was subjected to silica gel columnchromatography (chloroform/methanol=99/1) to give 52.6 mg (yield 80%) ofCompound II-81.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.259(3H, d, J=6.6 Hz), 1.266 (9H, d, J=6.6Hz), 1.993(1H, dd, J=5.0, 14.1 Hz), 2.131(3H, s), 2.881(2H, m),3.375(1H, dd, J=7.3, 14.1 Hz), 3.920(3H, s), 3.963(2H, s), 4.002(2H, s),4.953(1H, d, J=17.1 Hz), 5.016(1H, d, J=17.1 Hz), 7.098(1H, dd, J=5.0,7.3 Hz), 7.440-7.473(2H, m), 7.832(1H, d, J=8.1 Hz), 7.877(1H, d, J=8.8Hz), 7.959(1H, d, J=1.7 Hz), 8.592(1H, s), 9.139(1H, d, J=1.2 Hz)

FAB-MS (m/z): 643(M)⁺, 644 (M+1)⁺

EXAMPLE 44

Compound II-82

Substantially the same procedure as in Example 43 was repeated using51.9 mg (0.0958 mmol) of Compound (AA), 0.17 ml (1.9 mmol) of1-propanethiol, and 0.03 ml (0.2 mmol) of trifluoroacetic anhydride togive 52.3 mg (yield 83%) of compound II-82.

¹ H-NMR (DMSO-d₆ δ (ppm): 0.944(3H, t, J=7.3 Hz), 0.951 (3H, t, J=7.3Hz), 1.557-1.656(4H, m), 1.995(1H, dd, J=4.8, 14.1 Hz), 2.132(3H, s),2.462(2H, t, J=7.3 Hz), 2.470(2H, t, J=7.3 Hz), 3.378(1H, dd, J=7.4,14.1 Hz), 3.921(3H, s), 3.957(2H, s), 4.951 (1H, d, J=17.1 Hz),5.013(1H, d, 17.1 Hz), 7.102 (1H, dd, J=4.8, 7.4 Hz), 7.430-7.462(2H,m), 7.836(1H, d, J=8.3 Hz), 7.880(1H, d, J=8.6 Hz), 7.942(1H, d, J=1.5Hz), 8.599(1H, s), 9.122(1H, d, J=1.5 Hz)

FAB-MS (m/z): 643 (M)⁺, 644 (M+1)⁺

Compound 45

Compound II-83

Substantially the same procedure as in Example 43 was repeated using49.4 mg (0.0937 mmol) of Compound (AA), 0.20 ml (1.9 mmol) of1-butanethiol, and 0.03 ml (0.2 mmol) of trifluoroacetic anhydride togive 51.7 mg (yield 82%) of Compound II-83.

¹ H-NMR (DMSO-d₆) δ (ppm): 0.865(3H, t, J=7.4 Hz), 0.877 (3H, t, J=7.4Hz), 1.328-1.409(4H, m), 1.535-1.600 (4H, m), 1.995(1H, dd, J-4.9, 14.1Hz), 2.132(3H, s), 2.480(2H, t, J=7.4 Hz), 2.491(2H, t, J=7.4 Hz),3.377(1H, dd, J=7.5, 14.1 Hz), 3.921(5H, s), 3.958 (2H, s), 4.952(1H, d,J=16.9 Hz), 4.997(1H, d, J=16.9 Hz), 6.314(1H, s), 7.101(1H, dd, J=4.9,7.4 Hz), 7.432-7.458(2H, m), 7.834(1H, d, J=8.4 Hz), 7.880(1H, d, J=8.7Hz), 7.942(1H, d, J=1.5 Hz), 8.599 (1H, s), 9.123(1H, d, J=1.4 Hz)

FAB-MS (m/z): 671 (M)⁺

EXAMPLE 46

Compound II-84

Compound (AA) (45.3 mg, 0.0860 mmol) was dissolved in a mixture of 0.2ml of methanol and 2 ml of chloroform, and then 20 mg (0.086 mmol) ofcamphorsulfonic acid was added thereto, followed by stirring at roomtemperature for 17 hours. A saturated aqueous solution of sodiumbicarbonate was added to the reaction mixture, and the mixture wasextracted with chloroform. The organic layer was washed with an aqueoussolution of sodium chloride and dried over sodium sulfate. The solventwas evaporated under reduced pressure, and the residue was subjected tosilica gel column chromatography (chloroform/methanol=99/1) to give 23.1mg (yield 48%) of Compound II-84.

¹ H-NMR (DMSO-d₆ δ (ppm): 2.010(1H, dd, J=4.9, 14.1 Hz), 2.142(3H, s),3.341(3H, s), 3.364(3H, s), 3.383(1H, dd, J=7.4, 14.1 Hz), 3.925(3H, s),4.583(2H, s), 4.622(2H, s), 4.982(1H, d, J=16.9 Hz), 5.033(1H, d, J=16.9Hz), 6.330(1H, s), 7.127(1H, dd, J=4.9, 7.4 Hz), 7.441-7.464(2H, m),7.872(1H, d, J=8.4 Hz), 7.917(1H, d, J=8.7 Hz), 7.972(1H, d, J=1.1 Hz),8.611 (1H, s), 9.165(1H, d, J=1.0 Hz)

FAB-MS (m/z): 555 (M)⁺

EXAMPLE 47

Compound II-85

Substantially the same procedure as in Example 46 was repeated using asolution of 51.3 mg (0.0973 mmol) of Compound (AA) in a mixture of 0.2ml of ethanol and 2 ml of chloroform to give 24.1 mg (yield 42%) ofCompound II-85.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.189(3H, t, J=7.0 Hz), 1.199 (3H, t, J=7.0Hz), 1.999(1H, dd, J=4.9, 14.1 Hz), 2.142(3H, s), 3.385(1H, dd, J=7.4,14.1 Hz), 3.547 (2H, q, J=7.0 Hz), 3.563(2H, q, J=7.0 Hz), 3.925(3H, s),4.622(2H, s), 4.661(2H, s), 4.980(1H, d, J=16.9 Hz), 5.032(1H, d, J=16.9Hz), 6.325(1H, s), 7.124(1H, dd, J=4.9, 7.4 Hz), 7.447-7.467(2H, m),7.864(1H, d, J=8.3 Hz), 7.911(1H, d, J=8.7 Hz), 8.602 (1H, s), 9.162(1H,d, J=1.0 Hz)

FAB-MS (m/z): 583 (M)⁺

EXAMPLE 48

Compound II-86

Compound (BB) (Japanese Published Unexamined Patent Application No.295588/88) (978 mg, 1.69 mmol) was dissolved in 70 ml of1,2-dichloroethane, and then 0.17 ml (3.8 mmol) of fuming nitric acidwas added dropwise thereto under ice-cooling, followed by stirring atroom temperature for 30 minutes. The reaction mixture was diluted withchloroform and a saturated aqueous solution of sodium bicarbonate wasadded thereto. Insoluble material was collected by filtration and dried.The filtrate was washed with an aqueous solution of sodium chloride anddried over sodium sulfate, and the solvent was evaporated under reducedpressure. The residue and the insoluble material were combined to give946 mg (yield 90%) of Compound (CC) as a crude product.

FAB-MS (m/z): 625 (M+1)⁺

Compound (CC) (640 mg, 1.03 mmol) was dissolved in 30 ml of1,2-dichloroethane, and then 0.3 ml (3.58 mmol) of 1,2-ethanedithiol and0.2 ml (2.0 mmol) of boron trifluoride ether complex were added dropwisethereto at 0° C., followed by stirring for 30 minutes. A saturatedaqueous solution of sodium bicarbonate was added to the reactionmixture, and the mixture was extracted with chloroform. The organiclayer was washed with an aqueous solution of sodium chloride and driedover magnesium sulfate. The solvent was evaporated under reducedpressure, and the residue was subjected to silica gel columnchromatography (chloroform) to give 579 mq (yield 81%) of Compound (DD).

FAB-MS (m/z): 701 (M+1)⁺

Compound (DD) (579 mg, 0.827 mmol) was dissolved in 56 ml ofN,N-dimethylformamide, and then 400 mg of palladium/carbon was addedthereto, followed by stirring at 60° C. in an atmosphere of hydrogen for2 hours. Insoluble material was filtered off and the solvent wasevaporated under reduced pressure from the filtrate. The residue wassubjected to silica gel column chromatography (chloroform/methanol=98/2)to give 193 mg (yield 35%) of Compound (EE).

FAB-MS (m/z): 671 (M+1)⁺

Compound (EE) (193 mg, 0.288 mmol) was dissolved in 10 ml of chloroform,and then 0.1 ml (0.7 mmol) of triethylamine and 0.2 ml (2.5 mmol) ofethyl isocyanate were added thereto, followed by stirring at roomtemperature for 20 hours. After water was added, the mixture wasextracted with chloroform. The organic layer was washed with an aqueoussolution of sodium chloride and dried over magnesium sulfate. Thesolvent was evaporated under reduced pressure, and the residue wassubjected to silica gel column chromatography (chloroform/methanol=96/4)to give 211 mg (yield 99%) of Compound (FF).

FAB-MS (m/z): 742 (M+1)⁺

Compound (FF) (211 mg, 0.285 mmol) was dissolved in a mixture of 6 ml ofethanol and 6 ml of chloroform, and then 171 mg (1.01 mmol) of silvernitrate was added thereto at 50° C., followed by stirring for 20minutes. After the completion of reaction, insoluble material wasfiltered off. The filtrate was washed with a saturated aqueous solutionof sodium bicarbonate and an aqueous solution of sodium chloride, anddried over sodium sulfate. The solvent was evaporated under reducedpressure, and the residue was subjected to silica gel columnchromatography (chloroform/methanol=97/3) to give 118 mg (yield 62%) ofCompound (GG).

FAB-MS (m/z): 666 (M+1)⁺

Compound (GG) (100 mg, 0.150 mmol) was dissolved in a mixture of 4.5 mlof chloroform and 0.72 ml of methanol, and then 8.7 mg (0.23 mmol) ofsodium borohydride was added thereto at 0° C., followed by stirring for45 minutes. The reaction mixture was poured into water and the mixturewas extracted with chloroform. The organic layer was washed with anaqueous solution of sodium chloride and dried over sodium sulfate. Thesolvent was evaporated under reduced pressure, and the residue wassubjected to silica gel column chromatography (chloroform/methanol=95/5)to give 101 mg (yield 100%) of Compound (HH).

FAB-MS (m/z): 668 (M+1)⁺

Compound (HH) (21.7 mg, 0.0325 mmol) was dissolved in a mixture of 1 mlof 1,2-dichloroethane and 0.3 ml of methanol, and then 6 μl (0.03 mmol)of a 5.1N methanolic solution of sodium methoxide was added thereto,followed by stirring for one hour. The reaction mixture was poured intowater and the mixture was extracted with a mixture of chloroform andmethanol (9/1). The organic layer was washed with an aqueous solution ofsodium chloride and dried over magnesium sulfate. The solvent wasevaporated under reduced pressure, and the residue was subjected tosilica gel column chromatography (chloroform/methanol=90/10) to give14.9 mg (yield 79%) of Compound II-86.

¹ H-NMR (DMSO d₆) δ (ppm): 1.097(3H, t, J=7.2 Hz), 1.968 (1H, dd, J=4.9,13.9 Hz), 2.113(3H, s), 3.170(2H, dq, J=5.6, 7.2 Hz), 3.359(1H, dd,J=7.3, 13.9 Hz), 3.915(3H, s), 4.664(2H, s), 4.887(1H, d, J=16.9 Hz),4.947(1H, d, J=16.9 Hz), 6.081(1H, t, J=5.6 Hz), 6.273(1H, s), 7.090(1H,dd, J=4.9, 7.3 Hz), 7.364 (1H, dd, J=2.0, 9.0 Hz), 7.455(1H, dd, J=1.3,8.5 Hz), 7.782(1H, d, J=9.0 Hz), 7.826(1H, d, J=8.5 Hz), 8.189(1H, d,J=2.0 Hz), 8.493(1H, s), 8.537(1H, s), 9.127(1H, d, J=1.3 Hz)

FAB-MS (m/z): 584 (M+1)⁺

EXAMPLE 49

Compound II-87

Substantially the same procedure as in Example 43 was repeated using29.8 mg (0.0511 mmol) of Compound II-86 and 0.14 ml (1.6 mmol) ofethanethiol to give 24.2 mg (yield 76%) of Compound II-87.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.097(3H, t, J=7.1 Hz), 1.230 (3H, t, J=7.3Hz), 1.982(1H, dd, J=5.0, 14.1 Hz), 2.111(3H, s), 2.487(2H, dq, J=5.6,7.1 Hz), 2.987 (2H, q, J=7.3 Hz), 3.362(1H, dd, J=7.5, 14.1 Hz),3.914(3H, s), 3.939(2H, s), 4.888(1H, d, J=17.2 Hz), 4.950(1H, d, J=17.2Hz), 6.083(1H, t, J=5.6 Hz), 6.285(1H, s), 7.083(1H, dd, J=5.0, 7.5 Hz),7.370(1H, dd, J=2.1, 9.0 Hz), 7.436(1H, dd, J=1.6, 8.5 Hz), 7.783(1H, d,J=9.0 Hz), 7.825(1H, d, J=8.5 Hz), 8.188(1H, d, J=2.1 Hz), 8.496(1H, s),8.532(1H, s), 9.116(1H, d, J=1.6 Hz)

FAB-MS(m/z): 627(M)⁺

EXAMPLE 50

Compound II-88

Compound (AA) (50.4 mg, 0.0956 mmol) was dissolved in 0.7 ml ofdichloromethane, and then 0.09 ml (0.56 mmol) of triethylsilane and 0.73ml (9.5 mmol) of trifluoroacetic acid were successively added theretounder ice-cooling, followed by stirring at room temperature for 10minutes. The reaction mixture was neutralized with a 1N aqueous solutionof sodium hydroxide and the mixture was extracted with chloroform. Theorganic layer was washed with an aqueous solution of sodium chloride anddried over magnesium sulfate. The solvent was evaporated under reducedpressure, and the residue was subjected to silica gel columnchromatography (chloroform/methanol=90/10) to give 20.7 mg (yield 44%)of Compound II-88.

¹ H-NMR (DMSO-d₆ δ (ppm): 1.963(1H, dd, J=4.9, 13.9 Hz), 2.116(3H, s),2.510(3H, s), 2.529(3H, s), 3.353(1H, dd, J=7.3, 13.9 Hz), 3.914(3H, s),4.955(1H, d, J=17.2 Hz), 5.007(1H, d, J=17.2 Hz), 6.273(1H, s),7.074(1H, dd, J=4.9, 7.3 Hz), 7.287-7.313(2H, m), 7.764(1H, d, J=8.3Hz), 7.808(1H, d, J=8.5 Hz), 7.828(1H, s), 8.575(1H, s), 9.006(1H, s)

FAB-MS (m/z): 496 (M+1)⁺

EXAMPLE 51

Compound II-89

Compound (AA) (4.3 g, 8.16 mmol) was dissolved in 215 ml ofdichloromethane, and then 12.1 ml (163 mmol) of ethanethiol and 2.5 ml(17.7 mmol) of trifluoroacetic anhydride were successively addedthereto, followed by stirring at room temperature for 12 hours. Asaturated aqueous solution of sodium bicarbonate was added to thereaction mixture, and the mixture was extracted with chloroform. Theorganic layer was washed with an aqueous solution of sodium chloride anddried over sodium sulfate. The solvent was evaporated under reducedpressure, and the residue was subjected to silica gel columnchromatography (ethyl acetate/toluene=3/7) to give 4 mg (yield 0.08%) ofCompound II-89.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.233(3H, t, J=7.3 Hz), 1.253 (3H, dd, J=7.6,8.3 Hz), 2.007(1H, dd, J=4.6, 14.2 Hz), 2.139(3H, s), 2.492(2H, q, J=7.3Hz), 2.622-2.710(1H, m), 2.788-2.877(1H, m), 3.384(1H, dd, J=7.4, 14.2Hz), 3.926(3H, s), 3.979(2H, s), 4.106(1H, d, J=12.9 Hz), 4.285(1H, d,J=12.9 Hz), 4.961(1H, d, J=17.9 Hz), 5.025(1H, d, J=17.9 Hz), 6.325(1H,s), 7.132(1H, dd, J=4.8, 7.4 Hz), 7.433-7.473(2H, m), 7.887(1H, d, J=8.6Hz), 7.902(1H, d, J=8.3 Hz), 8.625(1H, s), 9.147(1H, s)

FAB-MS (m/z): 632 (M+1)⁺

EXAMPLE 52

Compound II-90

Compound (JJ) (Japanese Published Unexamined Patent Application No.295588/88) (18.5 g, 30.5 mmol) was dissolved in a mixture of 900 ml ofchloroform and 145 ml of methanol, and then 3.42 g (90.4 mmol) of sodiumborohydride was added thereto under ice-cooling, followed by stirringfor 25 minutes. The reaction mixture was poured into ice water, andinsoluble material was collected by filtration, washed with water, anddried under reduced pressure. The insoluble material was dissolved in amixture of 555 ml of 1,2-dichloroethane and 185 ml of methanol, and then0.925 ml (4.72 mmol) of a 5.1N methanolic solution of sodium methoxidewas added thereto, followed by stirring for one hour and a half. Thereaction mixture was poured into water, and insoluble material wascollected by filtration, dried under reduced pressure, and subjected tosilica gel column chromatography (chloroform/methanol=8/2) to give 0.350g (yield 2.3%) of Compound II-90.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.909(1H, dd, J=4.9, 13.4 Hz), 2.148(3H, s),3.134(1H, dd, J=7.3, 13.4 Hz), 3.757 (1H, dd, J=6.1, 11.3 Hz), 3.831(1H,dd, J=5.5, 11.3 Hz), 4.662(2H, d, J=5.6 Hz), 4.704(2H, d, J=5.6 Hz),4.944(1H, d, J=17.0 Hz), 5.007(1H, d, J=17.0 Hz), 5.098(1H, dd, J=5.5,6.1 Hz), 5.123(1H, t, J=5.6 Hz), 5.189(1H, t, J=5.6 Hz), 5.346(1H, s),6.942(1H, dd, J-4.9, 7.3 Hz), 7.398-7.459(2H, m), 7.722(1H, d, J=8.3Hz), 7.911(1H, d, J=8.8 Hz), 7.952 (1H, d, J=0.97 Hz), 8.538(1H, s),9.129(1H, m)

FAB-MS (m/z): 499 (M)⁺, 500 (M+1)⁺

EXAMPLE 53

Compound II-91

Compound (DD) (18.6 mg, 0.0266 mmol) was dissolved in a mixture of 1.5ml of 1,2-dichloroethane and 0.5 ml of methanol, and then 5 μl (0.026mmol) of a 5.1N methanolic solution of sodium methoxide was addedthereto, followed by stirring for one hour. The reaction mixture waspoured into water and the mixture was extracted with a mixture ofchloroform and methanol (9/1). The organic layer was washed with anaqueous solution of sodium chloride and dried over magnesium sulfate.The solvent was evaporated under reduced pressure, and the residue wassubjected to silica gel column chromatography (chloroform/methanol=95/5)to give 7.0 mg (yield 43%) of Compound II-91.

¹ H-NMR (DMSO-d₆) δ (ppm): 2.017(1H, dd, J=4.9, 14.4 Hz), 2.183(3H, s),3.408-3.452(2H, m), 3.588-3.651(2H, m), 3.940(3H, s), 5.122(1H, d,J=18.1 Hz), 5.175(1H, d, J=18.1 Hz), 5.943(1H, s), 6.549(1H, s),7.189(1H, dd, J=4.9, 7.3 Hz), 7.739(1H, dd, J=1.9, 8.7 Hz), 7.917(1H, d,J=8.7 Hz), 8.125(1H, d, J=9.4 Hz), 8.373(1H, dd, J=2.2, 9.4 Hz),8.733(1H, s), 8.848(1H, d, J=2.2 Hz), 9.353(1H, d, J=1.9 Hz)

FAB-MS (m/z): 617 (M+1)⁺

EXAMPLE 54

Compound II-92

Substantially the same procedure as in Example 53 was repeated using23.3 mg (0.0314 mmol) of Compound (FF) to give 14.7 mg (yield 71%) ofCompound II-92.

¹ H-NMR (DMSO-d₆) δ (ppm): 1.097(3H, t, J=7.1 Hz), 1.98 (1H, dd, J=4.9,14.0 Hz), 3.170(2H, dq, J=5.6, 12.7 Hz), 3.359(1H, dd, J=7.4, 14.0 Hz),3.401-3.464 (2H, m), 3.582-3.645(2H, m), 3.914(3H, s), 4.891 (1H, d,J=17.6 Hz), 4.956 (1H, d, J=17.6 Hz), 5.930 (1H, s), 6.081(1H, t, J=5.6Hz), 6.287(1H, s), 7.091 (1H, dd, J=4.9, 7.4 Hz), 7.379 (1H, dd, J=2.2,9.0 Hz), 7.683(1H, dd, J=1.7, 8.5 Hz), 7.783(1H, d, J=9.0 Hz), 7.850(1H,d, J=8.5 Hz), 8.183(1H, d, J=2.2 Hz), 8.499(1H, s), 8.534(1H, s),9.296(1H, d, J=1.7 Hz)

FAB-MS (m/z): 658 (M+1)⁺

While the invention has been set forth in considerable detail, theinvention disclosed herein is not to be limited to the actualdescription, but is to be afforded the full scope of the appended claimsand all equivalents thereto. Other embodiments are within the followingclaims.

What is claimed is:
 1. A method for enhancing the function ofcholinergic, striatal, basal forebrain and sensory neurons in a mammal,said method comprising administering to said mammal a therapeutic amountof a composition of the formula --N(CH₃)--W--N(CH₃)-- where represents aresidue of the formula: ##STR70## and W represents a radical of theformula --C(═Y)--NH--W¹ --NH--C(═Y)--, where W¹ is a hydrocarbyleneradical of 2 to 20 carbon atoms and Y is selected from the groupconsisting of O and S.
 2. The method of claim 1, wherein said sensoryneurons are dorsal root ganglion neurons.
 3. A method for protectingnerve cells from degeneration induced by excitatory amino acids, saidmethod comprising administering to a mammal a therapeutic amount of acomposition of the formula --N(CH₃)--W--N(CH₃)-- where represents aresidue of the formula: ##STR71## and W represents a radical of theformula --C(═Y)--NH--W¹ --NH--C(═Y)--, where W¹ is a hydrocarbyleneradical of 2 to 20 carbon atoms and Y is selected from the groupsconsisting of O and S.
 4. The method of claim 3 wherein saiddegeneration is associated with Alzheimer's disease.
 5. The method ofclaim 3 wherein said degeneration is associated with motor neurondisease.
 6. The method of claim 5 wherein said motor neuron disease isamyotrophic lateral sclerosis.
 7. The method of claim 3 wherein saiddegeneration is associated with Parkinson's disease.
 8. The method ofclaim 3 wherein said degeneration is associated with cerebrovasculardisease.
 9. The method of claim 8 wherein said cerebrovascular diseaseis ischemic.
 10. The method of claim 3 wherein said degeneration isassociated with AIDS dementia.
 11. The method of claim 3 wherein saiddegeneration is associated with epilepsy.
 12. The method of claim 3wherein said degeneration is associated with concussive injuries to thebrain.
 13. The method of claim 3 wherein said degeneration is associatedwith concussive injuries to the spinal cord.
 14. The method of claim 3wherein said degeneration is associated with penetrating injuries to thebrain.
 15. The method of claim 3 wherein said degeneration is associatedwith penetrating injuries to the spinal cord.
 16. The method of claim 3,wherein said degeneration is associated with Huntington's disease.
 17. Amethod for enhancing the function of a neuron in a mammal, wherein saidneuron is selected from the group consisting of sensory, cholinergic,basal forebrain, and striatal neurons, said method comprisingadministering to said mammal a therapeutic amount of a compoundrepresented by the formulae: ##STR72## wherein the followingsubstitutions are made:

    __________________________________________________________________________    Compound                                                                             R.sup.1        R.sup.2    X            R    Z.sup.2                    __________________________________________________________________________    II-1   H              H          CH.sub.2 N.sub.3                                                                           OH   H                          II-2   NHCONHC.sub.6 H.sub.5                                                                        H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-3   CH.sub.2 SOC.sub.2 H.sub.5                                                                   H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-4   H              H          CH.sub.2 OH  OCH.sub.3                                                                          H                          II-5   H              H          CONHC.sub.2 H.sub.5                                                                        OH   H                          II-6   H              H                                                                                         ##STR73##   OH   H                          II-7.sup.(2,7)                                                                       H              H          CH.sub.2 NHGly                                                                             OH   H                          II-8   H              H          CON(CH.sub.3).sub.2                                                                        OH   H                          II-9.sup.(3)                                                                         H              H          CH.sub.2 NHCO.sub.2                                                                             H                          II-10  Br             H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-11  H              H          CONH.sub.2   OH   H                          II-12  H              H          CH.sub.2 OH  OH   H                          II-13  H              H          CONHC.sub.3 H.sub.7                                                                        OH   H                          II-14.sup.(2)                                                                        H              H          CH.sub.2 NHSer                                                                             OH   H                          II-15  H              H          CH.sub.2 SOCH.sub.3                                                                        OH   H                          II-16  H              H          CHNOH        OH   H                          II-17  H              H          CON O        OH   H                          II-18.sup.(2,7)                                                                      H              H          CH.sub.2 NHPro                                                                             OH   H                          II-19  H              H          CHNNHC(NH)NH.sub.2                                                                         OH   H                          II-20  Br             Br         CO.sub.2 CH.sub.3                                                                          OH   O                          II-21  H              H          CONH(CH.sub.2).sub.2 OH                                                                    OH   H                          II-22  H              H          CO.sub.2 CH.sub.3                                                                          OH   O                          II-23  H              H          H            OH   H                          II-24  H              H          CHNNHCONH.sub.2                                                                            OH   H                          II-25  H              H          CH.sub.2 OCOCH.sub.3                                                                       OH   H                          II-26.sup.(3)                                                                        H              H          CH.sub.2 OC(CH.sub.3).sub.2 O                                                                   H                          II-29  NHCONHC.sub.2 H.sub.5                                                                        H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-30  CH.sub.2 SC.sub.2 H.sub.5                                                                    H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-31  Br             H          CH.sub.2 OH  OH   H                          II-32  Br             Br         CO.sub.2 CH.sub.3                                                                          OH   H                          II-33  CH.sub.2 SC.sub.6 H.sub.5                                                                    H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-34  Cl             Cl         CO.sub.2 CH.sub.3                                                                          OH   H                          II-36  H              H          CONHC.sub.6 H.sub.5                                                                        OH   H                          II-37  H              H                                                                                         ##STR74##   OH   H                          II-38  H              H          CH.sub.2 NHCO.sub.2 C.sub.6 H.sub.5                                                        OH   H                          II-39  NHCONHC.sub.2 H.sub.5                                                                        NHCONHC.sub.2 H.sub.5                                                                    CO.sub.2 CH.sub.3                                                                          OH   H                          II-40  N(CH.sub.3).sub.2                                                                            H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-41  CH.sub.3       H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-42  CH.sub.2 OCONHC.sub.2 H.sub.5                                                                H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-43  NHCO.sub.2 CH.sub.3                                                                          H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-44  Br             Br         CH.sub.2 OH  OH   H                          II-45  Br             Br         CONHC.sub.6 H.sub.5                                                                        OH   H                          II-46  Br             Br         CONHCH.sub.2 CH.sub.2 OH                                                                   OH   H                          II-47  CH.sub.2 OC.sub.2 H.sub.5                                                                    H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-48  CH.sub.2 N(CH.sub.3).sub.2                                                                   H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-49  CH.sub.2 SO.sub.2 C.sub.2 H.sub.5                                                            H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-50                                                                                 ##STR75##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-51  CH.sub.2 SC.sub.2 H.sub.5                                                                    CH.sub.2 SC.sub.2 H.sub.5                                                                CO.sub.2 CH.sub.3                                                                          OH   H                          II-52                                                                                 ##STR76##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-53                                                                                 ##STR77##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-54                                                                                 ##STR78##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-55                                                                                 ##STR79##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-56  CH.sub.2 SC.sub.2 H.sub.5                                                                    CH.sub.2 OH                                                                              CO.sub.2 CH.sub.3                                                                          OH   H                          II-57  H              H          CH.sub.2 NHCO.sub.2 CH.sub.3                                                               OH   H                          II-58  Br             H          CONH.sub.2   OH   H                          II-59  H              H          CH.sub.2 SC.sub.6 H.sub.5                                                                  OH   H                          II-60  H              H                                                                                         ##STR80##   OH   H                          II-61  H              H          CH.sub.2 SOC.sub.6 H.sub.5                                                                 OH   H                          II-63  OH             H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-65  CH.sub.2 SCH.sub.2 CH.sub.2 N(CH.sub.3).sub.2                                                H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-66  H              H          CH.sub.2 NH.sub.2                                                                          OH   H                          II-67  H              H          CONHCH.sub.3 OH   H                          II-68                                                                                 ##STR81##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-69                                                                                 ##STR82##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-70                                                                                 ##STR83##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-71                                                                                 ##STR84##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-72  CH.sub.2 S(CH.sub.2).sub.2 NH.sub.2                                                          H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-73                                                                                 ##STR85##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-74  CHNNHC(NH)NH.sub.2                                                                           H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-75                                                                                 ##STR86##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-76                                                                                 ##STR87##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-77  CHNN(CH.sub.3).sub.2                                                                         H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-78                                                                                 ##STR88##     H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-79  CH.sub.2 S(CH.sub.2).sub.2 NH-n-C.sub.4 H.sub.9                                              H          CO.sub.2 CH.sub.3                                                                          OH   H                          II-80  CH.sub.2 S(CH.sub.2).sub.2 N(CH.sub.3).sub.2                                                 CH.sub.2 S(CH.sub.2).sub.2 N(CH.sub.3).sub.2                                             CO.sub.2 CH.sub.3                                                                          OH   H                          VI-1.sup.(12)                                                                        H              H          CO.sub.2 CH.sub.3                                                                          OH   H                          VI-2.sup.(13)                                                                        H              NH.sub.2   CO.sub.2 CH.sub.3                                                                          OH   H                          __________________________________________________________________________     .sup.(1) Z.sup.1 and Z.sup.2 are both hydrogen, or both are combined          together to represent oxygen, where indicated.                                .sup.(2) NHamino acid linkage is an amide bond through the carboxyl group     of the amino acid.                                                            .sup.(3) X and R are combined together to form the linking group.             .sup.(7) Compound is in the form of the hydrochloride.                        .sup.(12) R.sup.8 = NHCONHC.sub.2 H.sub.5.                                    .sup.(13) R.sup.8 = NH.sub.2.                                            


18. The method of claim 17, wherein said functional derivative isCompound II-3.
 19. The method of claim 17, wherein said functionalderivative is Compound II-20.
 20. The method of claim 17, wherein saidfunctional derivative is Compound II-30.
 21. The method of claim 17,wherein said functional derivative is Compound II-33.
 22. The method ofclaim 17, wherein said functional derivative is Compound II-38.
 23. Themethod of claim 17, wherein said functional derivative is CompoundII-49.
 24. The method of claim 17, wherein said functional derivative isCompound II-51.
 25. The method of claim 17, wherein said functionalderivative is Compound II-65.
 26. The method of claim 17, wherein saidfunctional derivative is Compound II-69.
 27. The method of claim 17,wherein said functional derivative is Compound II-72.
 28. The method ofclaim 17, wherein said functional derivative is Compound II-73.
 29. Themethod of claim 17, wherein said functional derivative is CompoundII-79.
 30. The method of claim 17, wherein said functional derivative isCompound II-80.
 31. The method of claim 17, wherein said functionalderivative is Compound VI-1.
 32. The method of claim 17, wherein saidfunctional derivative is Compound VI-2.
 33. The method of claim 17,wherein said neuron is a cholinergic neuron.
 34. The method of claim 17,wherein said sensory neuron is a dorsal root ganglion neuron, and saidcompound is represented by formula (II): ##STR89## wherein the followingsubstitutions are made:

    ______________________________________                                        Com-                                    Z.sup.1(2)                            pound.sup.(1)                                                                       R.sup.1     X               R     Z.sup.2                               ______________________________________                                        II-1  H           CH.sub.2 N.sub.3                                                                              OH    H                                     II-2  NHCONHC.sub.6 H.sub.5                                                                     CO.sub.2 CH.sub.3                                                                             OH    H                                     II-3  CH.sub.2 SOC.sub.2 H.sub.5                                                                CO.sub.2 CH.sub.3                                                                             OH    H                                     II-4  H           CH.sub.2 OH     OCH.sub.3                                                                           H                                     II-5  H           CONHC.sub.2 H.sub.5                                                                           OH    H                                     II-6  H                                                                                          ##STR90##      OH    H                                     II-8  H           CON(CH.sub.3).sub.2                                                                           OH    H                                     II-9.sup.(3)                                                                        H           CH.sub.2 NHCO.sub.2   H                                     II-10 Br          CO.sub.2 CH.sub.3                                                                             OH    H                                     II-11 H           CONH.sub.2      OH    H                                     II-12 H           CH.sub.2 OH     OH    H                                     II-13 H           CONHC.sub.3 H.sub.7                                                                           OH    H                                     II-15 H           CH.sub.2 SOCH.sub.3                                                                           OH    H                                     II-17 H           CON O           OH    H                                     II-19 H           CHNNHC(NH)NH.sub.2                                                                            OH    H                                     II-20.sup.(1)                                                                       Br          CO.sub.2 CH.sub.3                                                                             OH    O                                     II-21 H           CONH(CH.sub.2).sub.2 OH                                                                       OH    H                                     II-23 H           H               OH    H                                     II-24 H           CHNNHCONH.sub.2 OH    H                                     II-25 H           CH.sub.2 OCOCH.sub.3                                                                          OH    H                                     II-30 CH.sub.2 SC.sub.2 H.sub.5                                                                 CO.sub.2 CH.sub.3                                                                             OH    H                                     II-32 Br          CO.sub.2 CH.sub.3                                                                             OH    H                                     ______________________________________                                         .sup.(1) R.sup.2 is hydrogen, except in compound II20 and II32 where          R.sup.2 = Br.                                                                 .sup.(2) Z.sup.1 and Z.sup.2 are both hydrogen, or both are combined          together to represent oxygen, where indicated.                                .sup.(3) X and R are combined together to form the linking group.        


35. The method of claim 1 wherein said composition is administered inconjunction with a trophic factor.
 36. The method of claim 3, whereinsaid composition is administered in conjunction with a trophic factor.37. The method of claim 17 wherein said functional derivative isadministered in conjunction with a trophic factor.
 38. The method ofclaim 34 wherein said functional derivative is administered inconjunction with a trophic factor.
 39. The method of any one of claims35-38, wherein said trophic factor is a member of the neurotrophinfamily.
 40. The method of claim 39 wherein said member of theneurotrophin family is nerve growth factor (NGF).
 41. The method ofclaim 17, wherein said neuron is a cholinergic neuron, and saidfunctional derivative is represented by the formula (II): ##STR91##wherein R¹ and R² are H; X is CO₂ CH₃ ; R is OH; and Z¹ and Z² are eachH.
 42. The method of claim 17, wherein said neuron is a striatal neuron,and said compound is represented by formula (II): ##STR92## wherein thefollowing substitutions are made:

    ______________________________________                                        Com-                                      Z.sup.2(1)                          pound R.sup.1     R.sup.2                                                                             X            R    Z.sup.1                             ______________________________________                                        II-1  H           H     CH.sub.2 N.sub.3                                                                           OH   H                                   II-35 H           H     CO.sub.2 n-C.sub.6 H.sub.13                                                                OH   H                                   II-20 Br          Br    CO.sub.2 CH.sub.3                                                                          OH   O                                   II-10 Br          H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-28 O-n-C.sub.3 H.sub.7                                                                       H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-5  H           H     CONHC.sub.2 H.sub.5                                                                        OH   H                                   II-29 NHCONHC.sub.2 H.sub.5                                                                     H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-2  NHCONHC.sub.6 H.sub.5                                                                     H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-3  CH.sub.2 SOC.sub.2 H.sub.5                                                                H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-30 CH.sub.2 SC.sub.2 H.sub.5                                                                 H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-6  H           H                                                                                    ##STR93##   OH   H                                   II-31 Br          H     CH.sub.2 OH  OH   H                                   II-32 Br          Br    CO.sub.2 CH.sub.3                                                                          OH   H                                   II-33 CH.sub.2 SC.sub.6 H.sub.5                                                                 H     CO.sub.2 CH.sub.3                                                                          OH   H                                   II-34 Cl          Cl    CO.sub.2 CH.sub.3                                                                          OH   H                                   ______________________________________                                         .sup.(1) Z.sup.1 and Z.sup.2 are both hydrogen, or both are combined          together to represent oxygen, where indicated.                                .sup.(2) R.sup.3 is CH.sub.2CHCH.sub.2 ; R.sup.4 is H.                   


43. The method of claim 17, wherein said neuron is a basal forebrainneuron, and said functional derivative is represented by the formula II:##STR94## wherein the following substitutions are made:

    __________________________________________________________________________    Compound                                                                             R.sup.1     R.sup.2                                                                              X        R   Z.sup.2, Z.sup.1                       __________________________________________________________________________           H           H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-3   CH.sub.2 SOC.sub.2 H.sub.5                                                                H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-5   H           H      CONHC.sub.2 H.sub.5                                                                    OH  H                                      II-10  Br          H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-20  Br          Br     CO.sub.2 CH.sub.3                                                                      OH  O                                      II-21  H           H      CONH(CH.sub.2).sub.2 OH                                                                OH  H                                      II-22  H           H      CO.sub.2 CH.sub.3                                                                      OH  O                                      II-30  CH.sub.2 SC.sub.2 H.sub.5                                                                 H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-32  Br          Br     CO.sub.2 CH.sub.3                                                                      OH  H                                      II-51  CH.sub.2 SC.sub.2 H.sub.5                                                                 CH.sub.2 SC.sub.2 H.sub.5                                                            CO.sub.2 CH.sub.3                                                                      OH  H                                      II-62  H           H      CO.sub.2 n-hexyl                                                                       OH  H                                      II-63  OH          H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-64  O n-propyl  H      CO.sub.2 CH.sub.3                                                                      OH  H                                      II-65  CH.sub.2 SCH.sub.2 CH.sub.2 N(CH.sub.3).sub.2                                             H      CO.sub.2 CH.sub.3                                                                      OH  H                                      __________________________________________________________________________     .sup.(1) Z.sup.1 and Z.sup.2 are both hydrogen, or both are combined          together to represent oxygen, where indicated.                           